Colonization of Zea Mays by the Nitrogen Fixing Bacterium Gluconacetobacter Diazotrophicus

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Colonization of Zea mays by the nitrogen fixing bacterium Gluconacetobacter diazotrophicus

Nikita Eskin The University of Western Ontario

Supervisor Dr. Lining Tian The University of Western Ontario

Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Nikita Eskin 2012

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COLONIZATION OF ZEA MAYS BY THE NITROGEN FIXING BACTERIUM GLUCONACETOBACTER DIAZOTROPHICUS

(Spine title: Colonization of Zea mays by Gluconacetobacter diazotrophicus) (Thesis format: Monograph)

Nikita Eskin

Graduate Program in Biology

A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science

The School of Graduate and Postdoctoral Studies Western University London, Ontario, Canada

THE UNIVERSITY OF WESTERN ONTARIO SCHOOL OF GRADUATE AND POSTDOCTORAL STUDIES

CERTIFICATE OF EXAMINATION

Supervisor Examiners

______Dr. Lining Tian Dr. Marc-André Lachance

______Co-Supervisor Dr. Greg Thorn

______Dr. Hugh Henry Dr. Richard B. Gardiner

Supervisory Committee

______Dr. Brian McGarvey

______Dr. Marc-André Lachance

The thesis by

Nikita Eskin

entitled:

Colonization of Zea mays by the nitrogen fixing bacterium Gluconacetobacter diazotrophicus

is accepted in partial fulfilment of the requirements for the degree of Master of Science

Date______Chair of the Thesis Examination Board ii

Abstract

Gluconacetobacter diazotrophicus, an endophytic nitrogen-fixing bacterium, is capable of supplying its host plant sugarcane with significant amounts of nitrogen. The objectives of this study were to investigate potential correlations between sucrose content in corn and colonization of G. diazotrophicus and to determine the effectiveness of soil drench, root dip, and aseptic methods of inoculation. The bacterium was detected in all seven corn genotypes containing different levels of sucrose with the aseptic method of inoculation and had an inoculation efficiency of 93%. Colonization was not detected within the corn genotypes using the soil drench and root dip methods of inoculation under greenhouse conditions. No nitrogenase activity was detected within colonized corn genotypes when analyzed by an acetylene reduction assay. This study indicated that the method of inoculation was a greater factor associated with G. diazotrophicus colonization than the sucrose content within the corn genotypes.

Keywords: colonization, corn, inoculation, genotype, Gluconacetobacter diazotrophicus

iii

Acknowledgements

I would like to sincerely thank my supervisor Dr. Lining Tian for his support and guidance throughout the past few years, and for the intellectually stimulating and challenging environment that he provided. I would like to thank my co-supervisor Dr. Hugh Henry for his guidance and for being supportive and available whenever research needed to be discussed. I thank my advisory committee: Dr. Brian McGarvey, for his critical editing of my thesis and for his technical assistance and input, and Dr. Marc-Andre Lachance for his sound advice and helpful suggestions.

I extend a special thank you to the lab technicians and staff from Agriculture and

Agri-Food Canada for all of their insightful input and technical support: Lisa Amyot, Ted

Blazejowski, Melinda Demendi, Mimmie Lu, Bob Pocs, and Sukhminder Sawhney. I am grateful towards my fellow lab mates and friends who were always there to either help with an experiment or to just go for a coffee: Mehran Dastmalchi, Korey Kilpatrick, Danny Kim,

Robin Lou, Eridan Pereira, Jessie Wong, Patrick Yoon, and Vanessa Yoon.

I am thankful for Agriculture Environmental Renewal Canada Inc., NSERC, and

Grain Farmers of Ontario for their support and for funding my research.

Most importantly, I would like to thank my family, whose help, support, and guidance have made me what I am today. Lastly, I thank Rachel Lafleche, as without her love, devotion, and patience I would not have had the strength and motivation to accomplish all that I have.

Table of Contents

Certificate of Examination ii

Abstract and Keywords iii

Acknowledgements iv

Table of Contents v

List of Tables vii

List of Figures viii

List of Appendices ix

List of Abbreviations xi

Chapter 1. Introduction 1

1.1 Corn production 1 1.2 Corn prices 2 1.3 Nitrogen 3 1.3.1 Nitrogen in agriculture 3 1.3.2 Nitrogen fertilizers 3 1.3.3 Nitrogen pollution 5 1.4 Biological nitrogen fixation 7 1.4.1 Biological nitrogen fixation in agriculture 7 1.4.2 Rhizobia 8 1.4.3 Non-specific nitrogen fixation 9 1.5 Gluconacetobacter diazotrophicus 10 1.5.1 Discovery and taxonomy 10 1.5.2 Characteristics of G. diazotrophicus 13 1.5.3 Nitrogenase enzyme 15 1.5.4 Auxin production 17 1.5.5 Endophytic localization 17 1.5.6 Recent studies of G. diazotrophicus 18 1.6 Rationale, hypothesis and objectives 20

Chapter 2. Materials and Methods 22

2.1 Culturing of G. diazotrophicus 22 2.2 Identification by polymerase chain reaction 22 2.3 Corn and sorghum genotypes 25

2.4 Greenhouse trials 27 2.4.1 Planting 27 2.4.2 Greenhouse inoculation 28 2.4.2.1 Soil drench 28 2.4.2.2 Root dip 28 2.4.3 Harvesting 30 2.5 Aseptic trials 30 2.5.1 Germination 30 2.5.2 Planting and inoculating 31 2.5.3 Harvesting 31 2.6 DNA extraction 32 2.7 Acetylene reduction assay 34 2.8 Sucrose analysis 35

Chapter 3. Results 37

3.1 Confirmational analysis 37 3.1.1 Bacterial growth 37 3.1.2 PCR confirmation 37 3.1.3 Nitrogenase activity confirmation 39 3.2 Green house trials 42 3.2.1 Soil drench trials 42 3.2.2 Root dip trials 46 3.3 Aseptic trials 46 3.4 Acetylene reduction assay analysis 48 3.5 Sucrose analysis 48

Chapter 4. Discussion and Conclusion 54

4.1 Laboratory grown cultures of G. diazotrophicus 54 4.2 PCR verification 54 4.3 Nitrogenase activity of G. diazotrophicus 55 4.4 Gluconacetobacter diazotrophicus colonization under greenhouse conditions 57 4.5 Gluconacetobacter diazotrophicus colonization under aseptic conditions 59 4.6 Nitrogenase activity within colonized corn 60 4.7 Effect of sucrose on G. diazotrophicus colonization 61 4.8 Conclusions 63

References 65

Appendix 72

Curriculum Vitae 95

List of Tables

Table 1.1 Corn growth nutrients and their corresponding deficiency symptoms 4

Table 1.2 Natural crop habitats of G. diazotrophicus 12

Table 1.3 Key characteristics of G. diazotrophicus 14

Table 2.1 PCR primer information for the detection of corn and sorghum ubiquitin and G. diazotrophicus 23

Table 2.2 General information regarding corn and sorghum genotypes 26

Table 2.3 Pro Mix Components 29

Table 2.4 Components of the DNA extraction buffer 33

Table 3.1 Nitrogenase activity of G. diazotrophicus measured by ARA 43

Table 3.2 Analysis of bacterial presence in soil drench inoculated samples of greenhouse sorghum via PCR 45

Table 3.3 Analysis of bacterial presence in soil drench inoculated control samples of greenhouse sorghum via PCR 45

Table 3.4 Analysis of bacterial presence in root dip inoculated samples of greenhouse sorghum via PCR 47

Table 3.5 Analysis of bacterial presence in root dip inoculated control samples of greenhouse sorghum via PCR 47

Table 3.6 Analysis of bacterial presence in aseptically inoculated corn via PCR 49

Table 3.7 Analysis of bacterial presence in aseptically inoculated control corn via PC 49

Table 3.8 ARA analysis of aseptically inoculated corn 50

Table 3.9 ARA analysis of soil drench inoculated sorghum genotype N111 50

Table 3.10 ARA analysis of root dip inoculated sorghum genotype N111 50

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List of Figures

Figure 3.1 Morphology of G. diazotrophicus in various LGIP media supplemented with 10 mM NH4(SO4)2 38 Figure 3.2 Nested PCR amplification of G. diazotrophicus Pal5 strain 40 Figure 3.3 Nested PCR sensitivity for Pal5 strain 40 Figure 3.4 PCR amplification of ubiquitin 40 Figure 3.5 Ethylene standard Curves 41 Figure 3.6 Sucrose concentrations in the roots of seven corn genotypes 52 Figure 3.7 Sucrose concentrations in the stems of seven corn genotypes 52 Figure 3.8 Sucrose concentrations in the leaves of seven corn genotypes 53

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List of Appendices

A.1 LGIP medium 72

A.2 Analysis of bacterial presence in soil drench inoculated root samples of greenhouse corn via PCR 73

A.3 Analysis of bacterial presence in soil drench inoculated stem samples of greenhouse corn via PCR 73

A.4 Analysis of bacterial presence in soil drench inoculated leaf samples of greenhouse corn via PCR 74

A.5 Analysis of bacterial presence in soil drench inoculated control root samples of greenhouse corn via PCR 74

A.6 Analysis of bacterial presence in soil drench inoculated control stem samples of greenhouse corn via PCR 75

A.7 Analysis of bacterial presence in soil drench inoculated control leaf samples of greenhouse corn via PCR 75

A.8 Root tissue PCR analysis of soil drench inoculated sorghum genotype N111 76

A.9 Stem tissue PCR analysis of soil drench inoculated sorghum genotype N111 77

A.10 Leaf tissue PCR analysis of soil drench inoculated sorghum genotype N111 78

A.11 Trial 1 analysis of bacterial presence in root dip inoculated root samples of greenhouse corn via PCR 79

A.12 Trial 1 analysis of bacterial presence in root dip inoculated stem samples of greenhouse corn via PCR 79

A.13 Trial 1 analysis of bacterial presence in root dip inoculated leaf samples of greenhouse corn via PCR 80

A.14 Trial 1 analysis of bacterial presence in root dip inoculated control root samples of greenhouse corn via PCR 80

A.15 Trial 1 analysis of bacterial presence in root dip inoculated control stem samples of greenhouse corn via PCR 81

A.16 Trial 1 analysis of bacterial presence in root dip inoculated control leaf samples of greenhouse corn via PCR 81

A.17 Trial 2 analysis of bacterial presence in root dip inoculated root samples of greenhouse corn via PCR 82

A.18 Trial 2 analysis of bacterial presence in root dip inoculated stem samples of greenhouse corn via PCR 82

A.19 Trial 2 analysis of bacterial presence in root dip inoculated leaf samples of greenhouse corn via PCR 83

A.20 Trial 2 analysis of bacterial presence in root dip inoculated control root samples of greenhouse corn via PCR 83

A.21 Trial 2 analysis of bacterial presence in root dip inoculated control stem samples of greenhouse corn via PCR 84

A.22 Trial 2 analysis of bacterial presence in root dip inoculated control leaf samples of greenhouse corn via PCR 84

A.23 Root tissue PCR analysis of root dip inoculated sorghum genotype N111 85

A.24 Stem tissue PCR analysis of root dip inoculated sorghum genotype N111 86

A.25 Leaf tissue PCR analysis of root dip inoculated sorghum genotype N111 87

A.26 PCR analysis of aseptically inoculated corn genotype C0103 88 A.27 PCR analysis of aseptically inoculated corn genotype C0348 89 A.28 PCR analysis of aseptically inoculated corn genotype C0444 90 A.29 PCR analysis of aseptically inoculated corn genotype C0258 91 A.30 PCR analysis of aseptically inoculated corn genotype C0428 92 A.31 PCR analysis of aseptically inoculated corn genotype NSS120 93 A.32 PCR analysis of aseptically inoculated corn genotype UT128B 94

List of Abbreviations

AAFC Agriculture and Agri-Food Canada

ADP Adenosine diphosphate

ATP Adenosine triphosphate

ARA Acetylene reduction assay bp Base pair(s)

BNF Biological nitrogen fixation bu Bushel(s) cm Centimeter(s)

CFU Colony forming unit(s)

DDGS Distillers dried grains with solubles

ºC Degrees Celsius

DNA Deoxyribonucleic acid dNTP Deoxyribonucleic triphosphates

DP Dual purpose e- Electron

FID Flame ionized detector g Gram(s)

GC Gas chromatography

GUS β-glucuronidase h Hour(s) ha Hectare(s)

H+ Hydron

IAA Indole-3-acetic acid

Kg Kilogram(s)

L Liter(s) m/z mass over charge m Meter(s) mg Milligram(s) mL Milliliter(s) mm Millimeter(s) mM Millimolar(s) ms Milliseconds min Minute(s)

Mo-nitrogenase Molybdenum-dependent nitrogenase

MOX Methoxyamine

MS Mass spectrometry

MSD Mass selective detector

MSTFA N-methyl-N-(trimethylsilyl) trifluoroacetamide

Pi Phosphate

PCR Polymerase chain reaction

PQQ-GDH Pyrroloquinoline quinone-linked glucose dehydrogenase rDNA Ribosomal deoxyribonucleic acid

RNA Ribonucleic acid rpm Rotations per minute s Second(s)

SD Standard deviation

SE Standard error

µg Microgram(s) xii

µL Microliter(s)

UV Ultraviolet

VAM Vesicular arbuscular mycorrhiza vol Volume v/v Volume/volume

V Volt(s) w/v Weight/volume

WDG Wet distillers grain

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Chapter 1. Introduction

1.1 Corn production

In North America, Zea mays (corn) production is very important to the agricultural industry. The United States is the world’s leader in grain corn production, annually growing approximately 38% (316 million tons) of corn produced globally (USDA, 2012; IGC, 2012).

Ranked as one of the top ten grain corn producing nations, Canada annually produces approximately 1.2% (11.7 million tons) of the corn produced globally (USDA, 2012b).

Within Canada, Ontario produces the most corn, both grain and sweet, 7,747,400 tons and

112,771 tons respectively (Statistics Canada, 2011; OMAFRA, 2011). Grain corn produced within Canada and the United States is mainly used as livestock feed, but is also used in a wide range of food and industrial products (USGC, 2010). As livestock feed, corn is the main component in a mixture of grains which also includes oats, barley, and sorghum (USDA,

2009). Food and industrial usage of grain corn requires an initial process of either wet or dry milling depending on the desired product. Some examples of corn products resulting from either wet or dry milling include high-fructose corn syrup, starch, corn oil, cereal, corn flour, and ethanol fuel (USDA, 2009). Additionally, the by-products of ethanol fuel production including, both distillers dried grains with solubles (DDGS) and wet distillers grains (WDG), can also be used as livestock feed. Regarding the DDGS, up to 309 kg can be recovered as a by-product to be used as feed from every ton of grain corn used for ethanol fuel

(Bonnardeaux, 2007).

1.2 Corn prices

Corn production has greatly increased over time due to continual improvements in technology and production practices, which have also led to increased prices (USDA, 2009).

The price per ton of grain corn has almost doubled over the last 30 years, going up from

115$/ton to 207$/ton (OMAFRA, 2011b). Many factors have contributed to the rising costs of corn. These include poor yields from other parts of world, export restrictions of other crops, panic buying, hording, and a shrinking US dollar (Epp, 2012). However, one of the key factors responsible for the rising corn costs was the rising price of oil. Both corn production and transportation costs rose along with rising oil prices (Epp, 2012).

Furthermore, other sectors which rely on grain corn for livestock feed and as components in food and industrial products could be affected by these record high prices, which could in turn affect the consumers.

The increasing cost of grain corn can also be attributed to the increase in use and skyrocketing prices of nitrogen fertilizers. Over the last 40 years, the amount of fertilizers applied to corn crops has almost doubled (USDA, 2012c). One of the main reasons for the increase is the correlation between yield and the amount of fertilizer applied (Below and

Brandau, 2001). Additionally, most farmers over-fertilize their fields as a means of protection and insurance against possible nitrogen losses to ensure maximum attainable yields (Below and Brandau, 2001; Paulson and Babcock, 2010). The increase in use is also coupled with an increase in cost. Prices for nitrogen fertilizers have doubled for most forms and in certain cases have even tripled over the last 10 years (USDA, 2012c).

1.3 Nitrogen

1.3.1 Nitrogen in agriculture

For plants nitrogen is necessary as a primary constituent of nucleotides, proteins, and chlorophyll (Robertson and Vitousek, 2009). However, plants can only assimilate several forms of nitrogen, including ammonium, nitrates, and organic compounds (urea). The availability of fixed nitrogen (nitrate or ammonium converted from dinitrogen) is seen by many as the most yield-limiting factor related to the agricultural production of corn

(Muthukumarasamy et al., 2002). To achieve maximum yields of corn, a rate of 0.5 kg of nitrogen per bushel is commonly applied, which can lead to farmers adding between 168-336 kg of nitrogen per hectare planted (Below and Brandau, 2001). Although nitrogen is found in high abundance in the atmosphere, biologically available nitrogen in terrestrial ecosystems is in short supply. As well, corn can remove up to 42 kg of nitrogen per hectare from the natural nitrogen pools in soil (Robertson and Vitousek, 2009). Without supplementing fields with external sources of nitrogen, corn yield and quality would be very low, and would unlikely be capable of meeting today’s current demand. In addition to nitrogen, corn plants require other macro and micronutrients to properly grow and attain full yield and high quality; these along with their related deficiency symptoms are listed in Table 1.1.

1.3.2 Nitrogen fertilizers

Throughout history (pre-industrial), three main methods of adding nitrogen to fields have been used: 1) organic wastes (human and animal waste, and crop residue), 2) crop rotations (nitrogen-fixing legumes), and 3) leguminous cover plants which were plowed

Table 1.1 Corn growth nutrients and their corresponding deficiency symptoms

Nutrient Deficiency Symptoms

Iron Prominent interveinal chlorosis/ or necrosis Veins are prominent over length of leaf

Pale green plants Nitrogen Chlorosis/ or necrosis advance from leaf tip along midrib

Dark green plants Phosphorus Dark yellow chlorosis along the leaf margins Purple color

Dark green plants Potassium Chlorosis along leaf margins developing to brown striping and necrosis

Green-yellow plants with dark yellow Magnesium interveinal chlorosis advancing to rust- brown necrosis

Sulfur Pale yellow plants Uniformly yellow leaves without necrosis

Pale green plants Zinc White to pale yellow bands in lower half of leaf which advance to pale brown or gray necrosis

(Adapted from UNL, 2009)

under as green manure (alfalfa and clover) (Smil, 2002). These methods benefited crops, but could only add approximately 57 kg of nitrogen per hectare, which is not enough to attain desired corn yields. The solution to achieving maximum crop yield with the supplementation of nitrogen fertilizers was achieved by Fritz Haber and Carl Bosch in the early 1900’s

(Erisman et al., 2008). In 1908 Haber successfully synthesized ammonium, and in 1913

Bosch was able to use what Haber discovered and commercialize it in the large scale production of ammonium (Smil, 2002; Erisman et al., 2008). The Haber-Bosch process synthesizes ammonium by reacting atmospheric dinitrogen with hydrogen at high pressures and temperatures in the presence of iron (Erisman et al, 2008). Since its commercialization, synthetic nitrogen fertilizer use has constantly been increasing. In the year 1950, approximately 2.75 million tons of synthetic fertilizer were used, this number increased to

63.75 million tons in the year 2000 and increased again to 100 million tons in the year 2008

(Smil, 2011). In correlation with the increase in use of synthetic nitrogen fertilizers, crop yield, specifically corn in the United States, has increased from 94 bu/ ha in 1950 to 380 bu/ ha in 2008 (USDA, 2009).

1.3.3 Nitrogen pollution

Assimilation of applied nitrogen fertilizer by crops such as corn is typically less than

50%, meaning that more than half of the applied fertilizer remains unutilized (Cassman et al.,

2002). Adding to this, the fact that nitrogen is mobile, reactive, and hard to contain makes it very vulnerable to losses due to denitrification, volatilization, and leaching (Smil, 1999;

Cassman et al., 2002; Robertson and Vitousek, 2009). Leached reactive forms of nitrogen are capable of causing widespread environmental effects and severe consequences to human

health (Vitousek et al., 1997; Wolfe and Patz, 2002). Some of the main detrimental effects to the environment due to the vast increase in the addition of synthetic nitrogen include: the acidification of soils, lakes, and streams, the eutrophication and hypoxia of coastal ecosystems, and the loss of biodiversity within both terrestrial and aquatic ecosystems

(Vitousek et al., 1997; Galloway et al., 2003; Robertson and Vitousek, 2009). One of the most noticeable detrimental effects of nitrogen in the environment is found in coastal waters.

The Gulf of Mexico, the Adriatic Sea, the Baltic Sea, and many other areas now contain continually enlarging ‘dead zones,’ which are areas of water that are hypoxic (O2 concentrations less than 2-3 mg/L) or anoxic (no O2) (Vitousek, 1997; Galloway et al., 2003).

The Gulf of Mexico contains one the largest examples of a hypoxic zone, measuring approximately 20,000 km2, derived from the intense agricultural practices surrounding the

Mississippi River, which drains into the Gulf of Mexico (Robertson and Vitousek, 2009).

Hypoxic zones develop due to the overenrinchment of coastal waters by excess nutrients, which occur due to runoff from agricultural fields. The excess nutrients and organic matter lead to eutrophication, resulting in an increase in algal growth. The subsequent decomposition of the algae by ocean dwelling bacteria leads to a decrease in the overall concentration of O2 in the water, resulting in an unfavourable and inhospitable environment to many deep water organisms (Galloway, 2003; Robertson and Vitousek, 2009). In addition to large fish kills, nitrogen pollution has been linked to having a negative impact on the biotic diversity of marine ecosystems (Galloway, 2003). In terrestrial ecosystems, the addition of nitrogen, a limiting nutrient, can decrease the overall biodiversity of an ecosystem by changing which species are dominant (Vitousek, 1997). Aside from damaging the environment, excess nitrogen leached into water supplies can have detrimental effects on

human health. According to the World Health Organization, 10 mg/L of nitrate-N is the maximum standard for safe drinking water. Within the United States, 20% of wells providing drinking water in agricultural settings have tested over the maximum nitrate-N contamination level, compared to only 3% in urban settings (Burow et al., 2010). Humans ingesting drinking water contaminated with high levels of nitrate-N are primarily susceptible to methemoglobinemia and N-nitroso-induced cancers (UNEP, 2007). Methemoglobinemia is a potentially fatal disorder which lowers oxygen carrying capacity. This occurs when nitrite ions enter the blood stream and inactivate hemoglobin by oxidizing its iron moiety (Wolf and

Patz, 2002). The increase in cancer incidences, specifically bladder and ovarian cancer, has been linked to nitrate contaminated drinking water (Weyer et al., 2001). Endogenously, nitrates are reduced to nitrites, and subsequent nitrosation reactions form highly carcinogenic

N-nitro compounds (Weyer et al., 2001).

1.4 Biological nitrogen fixation

1.4.1 Biological nitrogen fixation in agriculture

Along with having a large impact on the surrounding environment, nitrogen fertilizers are very expensive to a farming operation, as mentioned earlier. With costs of fertilizers doubling or tripling over the last decade, farmers have seen nitrogen fertilizers account for up to 15% of all production costs (Duffy, 2009). Therefore, farmers use crop rotation as a means to decrease the amount of nitrogen fertilizers that they need to apply during a growing season. Crop rotation is the practice of planting different crops within the same area over subsequent seasons. Crop rotation differs from the continuous monoculture practice (growing a single species repeatedly on the same plot of land) and also provides benefits to the

agricultural system in which it is used (Bullock, 1992). Some of the main benefits provided through crop rotation include the prevention of soil erosion, increased soil microorganism diversity, decreased pest prevalence, and increased field fertility (Bullock, 1992). The importance of field fertility in the process of growing corn is immense. As mentioned before, to achieve maximum yield, corn requires the addition of nitrogen fertilizers within the 168-

336 kg/ ha range. Soybean can leave behind approximately 70 kg/ ha of nitrogen in above ground residue. Therefore, when soybean is grown before corn, it is capable of providing the corn with approximately 20-40% of its required nitrogen, meaning that farmers need to apply less nitrogen fertilizers in comparison to corn grown following corn (Peoples et al., 1995;

Robertson and Vitousek, 2009). The reason that soybean is capable of providing nitrogen to future crops from within its tissues is biological nitrogen fixation (BNF). The process of BNF can be defined as the reduction of dinitrogen to ammonia by means of a prokaryote (Mylona et al., 1995). This process can be symbiotic and is considered to be a monospecific association which evolved over 60 million years ago (Hirsch, 2004; Geetanjali, 2006). BNF is accomplished by a wide variety of prokaryotes; some can accomplish this as free living organisms, while others require a symbiotic association with plants (Mylona et al., 1995).

1.4.2 Rhizobia

Approximately 80% all of BNF is accomplished through the symbiotic interaction between legumes, diverse angiosperms consisting of over 18,000 species, and α- proteobacteria in the order Rhizobiales, family Rhizobiaceae (Geetanjali, 2006). The interaction between the nitrogen fixing bacteria and host plant is considered to be mutualistic because both organisms benefit from one another. The bacteria provide biologically fixed

nitrogen which can be directly used by the host plant and in contrast to nitrogen fertilizers is less susceptible to volatilization, denitrification, and leaching (Geetanjali, 2006). In return, the host plant provides photosynthetic products, mainly glucose, sucrose, and organic acids

(Bergersen, 1971). Within the Rhizobiaceae family of bacteria, this exchange of nutrients with legumes occurs within specific structures called nodules (Geetanjali, 2006). Nodules are located on the roots of legume plants and in addition to facilitating nutrient exchange, also provide an oxygen limiting environment that is critical to the bacterium’s ability to fix nitrogen (Geetanjali, 2006). The formation of nodules begins with the bacterium’s detection of a suitable host’s root system with the help of chemoattractants released by the host. This is followed by a series of reciprocal molecular conversation signals between the bacterium and plant leading to changes in the transcriptional regulation of genes, structural changes, and eventually the formation of a root nodule (Geetanjali, 2006). It is the specificity of this reciprocal communication that determines the host range of the bacterium (Fisher and Long,

1992). It is due to this host-specific interaction that the bacterium responsible for the BNF observed within legumes cannot be naturally introduced to other crops such as corn, wheat, barely, or sorghum (Fisher and Long, 1992). Within soybean, BNF can in some cases provide the plants with enough nitrogen that no significant difference is observed when compared to others supplemented with nitrogen fertilizers (Alves et al. 2003).

1.4.3 Non-specific nitrogen fixation

As discussed earlier, the host-specific Rhizobiaceae family of bacteria are only one example of bacteria capable of BNF. Non-specific nitrogen-fixing bacteria also exist and have opened up the possibility of symbiotic nitrogen fixation in a wide array of monocot

crops, including corn (Peoples et al., 1995; Muthukumarasamy et al., 2002). The majority of non-specific nitrogen fixing bacteria are free-living, as saprobes (living on plant residues), endophytes (living within plants), and rhizobacteria (living in close association with plant roots) (Gothwal et al. 2008). The two main types that require associations with host plants are endophytes and rhizobacteria, which can be classified as plant growth promoting bacteria because they are beneficial to their host plants (Saharan and Nehra, 2011). Rhizobacteria reside within a plants rhizosphere, an area of influence around a plants root system (Gothwal et al. 2008). Within the rhizosphere, the rhizobacteria tend to live in close proximity to the roots and depending on the bacterial species can benefit the associated plant in several ways outside of providing a source of fixed nitrogen. A few examples include plant disease suppression, improved nutrient acquisition, and phytohormone production (Saharan and

Nehra, 2011). The rhizobacteria in turn receive carbon and sources of energy that are leached into the rhizosphere from the host plant’s roots, and are necessary for the survival of the bacteria (Saharan and Nehra, 2011). Bacterial endophytes are either obligate or facultative depending on their ability to survive outside their host plants. Endophytes, like rhizobacteria, are capable of providing a wide array of beneficial attributes to their host plants in return for carbon and sources of energy (Saharan and Nehra, 2011).

1.5 Gluconacetobacter diazotrophicus

1.5.1 Discovery and taxonomy

Few nitrogen fixing endophytes have had as much attention as Gluconacetobacter diazotrophicus. The importance of G. diazotrophicus was first recognized when it was discovered within sugarcane plants in Alagoas, Brazil in 1988 by Cavalcante and Dobereiner

(1988). The bacterium’s ability to provide its host sugarcane, a monocot, with large amounts of fixed nitrogen without the formation of nodules led to the recognition of its importance.

The non-nodulating, endophytic characteristic of the bacterium left researchers hopeful of its potential to inhabit other monocot crops, including corn (Triplett 1996). Since its discovery,

G. diazotrophicus has been naturally found to inhabit several other crops, including sweet potato, coffee, and pineapple (Table 1.2) (Paula et al., 1991; Jimenez-Salgado et al., 1997;

Tapia-Hernandez et al., 2000). Gluconacetobacter diazotrophicus was initially given the name Saccharobacter nitrocaptans by Cavalcante and Dobereiner (1988) due to the important differences that separated it from all possible related bacteria, based on Bergey’s

Manual of Systematic Bacteriology 1984. Further research by Gillis and colleagues (1989) based on the bacterium’s genomic, phenotypic, and chemotaxonomic evidence constituted the need to create a new species for the bacterium within the genus Acetobacter. Therefore, it was renamed Acetobacter diazotrophicus (Gillis et al., 1988). Additional 16S ribosomal

RNA analysis of the bacterium resulted in an additional name change to Gluconacetobacter diazotrophicus (Yamada et al., 1997). This bacterium is in the phylum Proteobacteria, the class Alpha Proteobacteria, the order Rhodospirillales, the family Acetobacteraceae, and genus Gluconacetobacter (Kersters et al., 2006).

Table 1.2 Natural crop habitats of G. diazotrophicus

Country Crop Isolation source Reference

Root, root hair, Cavalcante and Brazil Sugarcane stem, leaf Dobereiner, 1988

Dobereiner et al., Brazil Cameroon grass Root, stem 1988

Dobereiner et al., Brazil Sweet potato Root, stem tuber 1988

Root, rhizosphere, Jimenez-Salgado et Mexico Coffee stem al., 1997

Root, rhizosphere, Loganathan et al., India Finger millet stem 1999

Matiru and Kenya Tea Root Thomson, 1998

Tapia-Hernandez et Mexico Pineapple Root, stem, leaf al., 2000

Root, rhizosphere, Muthukumarasamy India and Korea Wetland rice stem et al., 2005

Matiru and Kenya Banana Rhizosphere Thomson, 1998

Brazil VAM spore Internal Paula et al., 1991

1.5.2 Characteristics of G. diazotrophiocus

Gluconacetobacter diazotrophicus is a Gram-negative, acid-tolerant, obligate aerobe with cells that are straight rods with rounded ends measuring about 0.7-0.9 µm by 1-2 µm

(Cavalcante and Dobereiner, 1988; Gillis et al., 1988). Cells have between 1-3 lateral or peritrichous flagella and when viewed under a microscope can appear as single, paired, or chainlike structures without the presence of an endospore. (Cavalcante and Dobereiner, 1988;

Gillis et al., 1988; Muthukumarasamy et al., 2002). Key characteristics of G. diazotrophicus are listed in Table 1.3. High sucrose concentrations (10%) are the best source of carbon for the bacterium’s growth, but glucose, fructose, and galactose can also be used (Cavalcante and

Dobereiner, 1988). However, as the bacterium is unable to transport or take up sucrose it secretes an extracellular enzyme called levansucrase, which hydrolyzes sucrose into glucose and fructose (Martinez-Fleites et al., 2005; Hernandez et al., 1995). This enzyme is critical for the survival of the bacterium, and can constitute over 70% of all secreted proteins by specific strains of G. diazotrophicus (Hernandez et al., 1995). It is with the aid of levansucrase that the bacterium can survive and grow in sucrose concentrations of 30%

(Cavalcante and Dobereiner, 1988). G. diazotrophicus also contains a pyrroloquinoline quinone-linked glucose dehydrogenase (PQQ-GDH), which oxidizes glucose into gluconic acid in the extracellular environment (Attwood et al., 1991; Galar and Boiardi, 1995). The production of gluconic acid, coupled with high tolerance to low pH levels (2.5), make the bacterium a strong candidate for the industrial production of gluconic acid, a chemical used in cleaning products (Attwood et al., 1991; Stephan et al., 1991). More importantly, the

PQQ-GDH, which is primarily synthesized under nitrogen fixing conditions, produces a large

Table 1.3 Key characteristics of G. diazotrophicus

Characteristic Gluconacetobacter diazotrophicus

Gram reaction -

Colonies on LGI-P plates Dark orange

pH tolerance < 2.5

Oxidase -

Catalase +

Nitrate reductase -

Nitrogen fixation +

Nitrogen fixation product Ammonium

IAA production +

Growth in presence of 30% D-glucose +

Growth in presence of 10% ethanol -

(Adapted from Cavalcante and Dobereiner, 1988 and Gillis et. al., 1989)

amount of energy for the bacterium. The increase of energy combined with the timing of the protein’s synthesis shows its importance in providing the bacterium with additional energy during nitrogen fixation, as there is a high energy demand associated with the conversion of dinitrogen by the nitrogenase (Galar and Boiardi, 1995).

1.5.3 Nitrogenase enzyme

The nitrogenase of G. diazotrophicus is a molybdenum-dependent system (Mo- nitrogenase) and is capable of providing its host with a substantial amount of fixed nitrogen

(Fisher and Newton, 2005). 15N-aided nitrogen balance studies have shown that certain genotypes of sugarcane are capable of having up to 200 kg N per hectare fixed for them by

G. diazotrophicus, meeting approximately half of the crop’s nitrogen needs without the application of additional fertilizers (Lima et al., 1987; Boddey et al., 2001). The conversion of dinitrogen to ammonia, as shown in Equation 1.1, is catalyzed by the nitrogenase, a two- component metalloenzyme (Fisher and Newton, 2005). Mo-nitrogenases are made up of two

Equation 1.1 Nitrogenase catalyzed reduction of N2 to NH3

+ - N2 + 8H + 8e + 16ATP  2NH3 + H2 + 16ADP + 16Pi

(Adapted from Rees and Howard, 2000)

component proteins, the Fe protein containing the ATP-binding sites and the MoFe protein containing the substrate binding sites (Rees and Howard, 2000). G. diazotrophicus component proteins are each synthesized from a set of highly conserved nitrogen fixation

(nif) structural genes, very similar to other members of the class Alpha Proteobacteria (Fisher and Newton, 2005; Bertalan et al., 2009). What makes G. diazotrophicus unique is that it does not contain a nitrate reductase protein (Cavalcante and Dobereiner, 1988). Without the nitrate reductase protein in the bacterium, the nitrogenase does not become inhibited by rising levels of nitrites (Trinchant and Rigaud, 1982; Cavalcante and Dobereiner, 1988).

Additionally, the nitrogenase of G. diazotrophicus is not completely inhibited by the addition of ammonium, meaning that the bacterium is capable of undergoing nitrogen fixation in crops that are supplemented with low amounts of ammonium-based nitrogen fertilizers

(Stephan et al., 1991; Fisher and Newton, 2005). One substrate that does switch off nitrogenase activity is oxygen. Oxygen inhibits nitrogenase activity on three different levels: repressing nitrogenase synthesis at the genetic level, causing irreversible damage to the Fe protein, and reversible inhibition of the enzyme due to oxygen pressure (Goldberg et al.,

1987; Reis and Dobereiner, 1998). While oxygen can inhibit the nitrogen fixing capabilities of the bacterium, it is needed to ensure that an adequate amount of energy is also produced, especially for the high-energy-demanding process of nitrogen fixation (Reis and Dobereiner,

1998). In order to have uninterrupted nitrogen fixation and produce the proper amount of energy to run the nitrogen fixation process a limited amount of environmental oxygen is required. Therefore, G. diazotrophicus uses the high sucrose concentrations (10%) within its sugarcane hosts to protect itself from both the presence of oxygen and from high levels of ammonium (Reis and Dobereiner, 1998).

1.5.4 Auxin production

Aside from nitrogen fixation, G. diazotrophicus provides its host plants with an additional growth promoting factor, indole-3-acetic acid (IAA) (Fuentes-Ramirez, 1993;

Saravanan et al., 2008). The production of IAA by the bacterium has been linked to its survival within sugarcane. Because sugarcane is primarily propagated through stem cuttings, a location in which the bacterium resides, it is important for the bacterium to promote rooting through the biosynthesis of IAA and improve the sugarcane cutting’s growth (Fuentes-

Ramirez, 1993). An additional explanation for the biosynthesis of IAA by the bacterium could be a result of a key characteristic of the plant hormone. Sugar is very important in the root formation of sugarcane cuttings, and IAA causes sugars to accumulate at the site of IAA biosynthesis (Altman and Wareing, 1975). Therefore, it is postulated that G. diazotrophicus biosynthesizes IAA in order to increase the accumulation of sucrose in its general vicinity, important to both its survival and the plant’s.

1.5.5 Endophytic localization

G. diazotrophicus, as mentioned earlier, is an obligate endophyte, with the exception of being capable of surviving within the spores of the vesicular-arbuscular mycorrhizal fungus Glomus clarum and within the root hairs of a host plants rhizosphere (Paula et al,

1991; Jimenez-Salgado et al., 1997; Muthukumarasamy et al, 2002). Within host plants, the bacterium primarily inhabits intercellular apoplastic spaces, the xylem, the xylem parenchyma, and intracellularly without nodulation (James et al., 2001; Cocking et al., 2006).

G. diazotrophicus is capable of entering its host plants through the roots, stems, and leaves

(James et al., 2001). With regard to the roots, the bacterium enters through spaces between

cells in the root meristem and at areas of lateral root emergence. Within the stem, the bacterium enters at breaks caused by the separation of plantlets into individuals. Lastly, within the leaves, the bacterium enters through damaged stomata (James et al., 2001). Once established within a host, G. diazotrophicus can grow up to 108 CFU per gram of sugarcane tissue (Reis et al., 1994).

1.5.6 Recent studies of G. diazotrophicus

In addition to plants which G. diazotrophicus naturally inhabits, a number of additional non-native plants have proved to be capable of hosting this bacterium. These include but are not limited to: arabidopsis (Arabidopsis thaliana), tomato (Lycopersicon esculentum), and more importantly corn (Riggs et al., 2001; Cocking et al., 2006; Tian et al.,

2009). Several different studies have observed the ability of G. diazotrophicus to colonize corn plants under field, greenhouse, and aseptic conditions. The bacterium is capable of inhabiting several corn genotypes through several different means of inoculation: seed coating, applications to the base of stems, and root dipping (Riggs et al., 2001; Cocking et al.,

2006; Tian et al., 2009). Some of these studies have shown that under both field and greenhouse conditions G. diazotrophicus is capable of enhancing corn productivity, resulting in an increased yield (Riggs et al., 2001). Other studies have proven through β-glucuronidase

(GUS)-labeling that in addition to being capable of intracellular colonization of the roots, the bacterium is capable of expressing nitrogenase genes within corn plants. Unfortunately, research has yet to show any nitrogen fixation by G. diazotrophicus within corn plants.

Furthermore, findings have shown that the bacterium is unable to reach the same colonization levels in corn as in sugarcane, growing to only 103 CFU/g compared to 108 CFU/g (Reis et

al., 1994; Tian et al., 2009). One key difference between sugarcane and corn which studies have focused on is the difference in their sucrose content. Some sugarcane genotypes are capable of producing up to 62% dry weight sucrose, while some grain corn genotypes produce less than 1% dry weight sucrose (Tian et al., 2009; Sachdeva et al., 2011). This discrepancy in sucrose content has led to the high apoplastic sucrose hypothesis (Riggs et al.,

2001). The high apoplastic sucrose hypothesis postulates that G. diazotrophicus colonization levels in corn are inhibited by the large diversity of bacterial endophytes already harbored within. In high sucrose varieties, however, the increased osmotic potential caused by increased apoplastic sucrose levels would inhibit those endophytes, allowing a larger population of G. diazotrophicus to grow (Riggs et al., 2001). Furthermore, other studies have suggested that the absence of nitrogen fixation is due to the small population size of G. diazotrophicus in corn (Tian et al., 2009). Although Cocking et al. (2006) demonstrated that the nitrogenase gene nifH is expressed within corn plants, no evidence supports the enzyme being activated. A possible explanation could be a lack of quorum sensing, a cell-to-cell signalling mechanism (Reading and Sperandia, 2006). Quorum sensing refers to the ability of a bacterium to respond to autoinducers, hormone-like molecules which are capable of altering gene expression at a critical threshold population (Reading and Sperandio, 2006).

While it is known that the nifH gene is expressed, there is no evidence that the remainder of genes responsible for a functional nitrogenase are active. Therefore, there is still no evidence to suggest that nitrogen fixation occurs within corn.

1.6 Rationale, hypothesis and objectives

This study will investigate the colonization efficiency of G. diazotrophicus across seven corn genotypes using three different methods of inoculation: root dip, soil drench, and aseptic inoculation. Among the seven corn genotypes there are three distinct types; grain corn

(C0258 and C0428), newly bred grain corn with assumed high sucrose content (C0103,

C0348, and C0444), and sweet corn (NSS120 and UT128B). Research by Tian et. al. (2009) showed that inoculation into the grain corn and sweet corn genotypes used in this study was possible through the root dip method of inoculation; no nitrogenase activity was detected in that study. The newly bred high sucrose content grain corn genotypes could be the missing key in attempts to achieve nitrogen fixation within corn successfully, as the genotypes could provide an adequate apoplastic sucrose environment for G. diazotrophicus to reach high colony numbers. Each corn genotype will be analyzed to determine the efficiency at which

G. diazotrophicus is capable of achieving successful colonization. Additionally, the different inoculation methods will be compared to determine which is most efficient at introducing the bacterium into its host. The root dip and soil drench methods of inoculation will include a sweet sorghum genotype as a methodological control, as recent results by Yoon (unpublished data) suggest high colonization efficiency rates. Root dip and aseptic inoculation trials have been attempted and have shown positive results of corn colonization by G. diazotrophicus.

Unfortunately those methods, while appropriate for greenhouse and small scale studies, are not feasible on a large scale farming operation. Therefore, soil drench, a new method for the inoculation of G. diazotrophicus into corn, will be attempted. This method, although untested with this bacterium, can be easily implemented into a large scale farming operation. Sucrose levels will be examined across all seven corn genotypes to determine their similarity to the

levels reported in sugarcane. Lastly, plants successfully inoculated with the bacterium will be tested to determine if G. diazotrophicus has an active nitrogenase within the corn plants.

As postulated in the apoplastic sucrose hypothesis, higher sucrose levels should lead to higher levels of G. diazotrophicus inoculation. Additionally, environments in which corn’s natural endophytic bacteria are absent would be more suitable for G. diazotrophicus inoculation. Therefore, it is hypothesized that (1) colonization efficiency in corn varieties will be affected by their sucrose content and (2) inoculation efficiency will be highest when corn is grown and inoculated under aseptic conditions.

The objectives of this study are (1) to determine which corn genotypes are the most suitable for hosting G. diazotrophicus, (2) to determine which method of inoculation is most efficient at introducing G. diazotrophicus into host plants, (3) to determine if nitrogenase is functional when G. diazotrophicus is colonized within corn, and (4) to determine if corn genotypes with the highest sucrose concentrations have the highest colonization efficiencies.

Chapter 2. Materials and Methods

2.1 Culturing of G. diazotrophicus

The G. diazotrophicus bacterial strain Pal5 was used in this study. The strain was kindly provided by Dr. Zhongmin Dong (Saint Mary’s University, Halifax, Nova Scotia,

Canada). The Pal5 strain (wild-type; culture collection: ATCC 49037), which is capable of

N2 fixation, was originally isolated from the roots of sugarcane plants in Alagoas, Brazil

(Cavalcante and Dobereiner, 1988).

The G. diazotrophicus PAL5 strain was cultured in LGIP medium (Appendix A.1).

Solid, semisolid, and liquid variations of the LGIP medium supplemented with 10 mM

NH4(SO4)2 were used in culturing. Incubation was carried out in the dark at a temperature of

28 ºC while the duration of incubation varied depending on the LGIP variants. Semi-solid and liquid cultures were incubated for two days; liquid cultures received constant agitation at

180 rpm. Plates were incubated for 4-5 days.

2.2 Identification by polymerase chain reaction

Identification of G. diazotrophicus was done by means of nested PCR. Primers used in identification were specifically designed based on the 16S rDNA of G. diazotrophicus, listed in Table (2.1). The nested PCR contained two rounds of amplification. The initial round of amplification used primers GDI25F and GDI923R (Tian et al., 2009) and resulted in an 899 bp amplicon which was then subjected to a second round of amplification with the use of the GDI39F and GDI916R primers (Franke-Whittle et al., 2005), which in turn produced

Table 2.1 PCR primer information for the detection of corn and sorghum ubiquitin and G. diazotrophicus

Primer Sequence Product size Reference

GDI25F 5’-TGAGTAACGCGTAGGGATCTG-3’ 899 bp (Tian et al., 2009) GDI923R 5’-GGAAACAGCCATCTCTGACTG-3’

GDI39F 5’-TAGTGGCGGACGGGTGAGTAACG-3’ 879 bp (Franke-Whittle et al., 2005) GDI916R 5’-CCTTGCGGGAAACAGCCATCTC-3’

Ub-U29162-R 5’-CCTTCTGAATGTTGTAATCCGCA-3’ 218 bp (Sorgona et al., 2011) Ub-U29162-F 5’-CCACTTGGTGCTGCGTCTTAG-3’

an 879 bp product. Both rounds of the nested PCR were subjected to analysis via gel electrophoresis.

A mastermix with a total volume of 20 µL was used for PCR analysis. The PCR mastermix consisted of 10× PCR buffer, 0.2 µL of each of the two primers, 0.2 mM of all

-1 four dNTPs, Taq-polymerase (5 U µL ), and sterile milli-Q H2O. For amplification of bacterial DNA, colonies that were grown for four days were picked from plates, and diluted in 100 µL of sterile milli-Q H2O, and subsequently 1 µL of the diluted solution was transferred into 200 µL PCR microtubes into which the previously mentioned mastermix was added. For experimental trials, a 1 µL aliquot of extracted DNA was used instead of a 1 µL aliquot of the diluted bacterial solution. All PCR amplifications were performed on either the

Eppendorf Mastercycler Pro S Vapo.Protect thermal cycler or the Eppendorf Mastercycler

EpGradient thermal cycler. The following temperature profile was used for the first round of the nested PCR: 35 cycles of denaturation for 45 s at 95 ºC, annealing for 45 s at 63 ºC, and extension for 60 s at 72 ºC. Following the completion of the first round a 1 µL aliquot of the amplified PCR product was used as the template for the second round of the nested PCR, which utilized the following temperature profile: 30 cycles of denaturation for 30 s at 95 ºC, annealing for 45 s at 62 ºC, and extension for 30 s at 72 ºC. Temperature profiles for both rounds of the PCR contained a 10 min denaturation step at 95 ºC at the beginning and a final

10 min extension at 72 ºC. Samples were then maintained at 4 ºC until removed.

Identification of corn and sorghum ubiquitin was accomplished through standard

PCR. Detailed information regarding primers Ub-U29162-R and Ub-U29162-F (Sorgona et al., 2011) are listed in Table (2.1). The following temperature profile was used to amplify the

218 bp ubiquitin product: 30 cycles of denaturation for 30 s at 94 ºC, annealing for 45 s at

50.2 ºC, and extension for 60 s at 72 ºC. The 30 cycles of the temperature profile began with an initial denaturation step of 3 min at 94 ºC and finished with an additional 7 min of extension at 72 ºC, which was followed by a temperature drop to 4 ºC until samples were removed. The PCR mastermix for ubiquitin amplification was the same as listed earlier for

G. diazotrophicus amplification.

The amplified products from the PCR were evaluated using gel electrophoresis. A 1% agarose gel was stained with 10 µl EtBr at a 500 µg mL-1 concentration onto which the PCR products were loaded. A running time of 40 min at 100 V was used. Visualization of the gels was accomplished with the BioRad Quantity One Gel Doc software (Version 4.4.1) with a

Foto/Prep UV transilluminator.

2.3 Corn and sorghum genotypes

Seven corn genotypes and one sorghum genotype were used in this study (Table 2.2).

Of the corn genotypes five were inbred grain corn varieties provided by Dr. Lana Reid from

AAFC Ottawa. Three of the five grain corn genotypes were newly bred and were presumed to have high sucrose content. The remaining two corn genotypes were sweet corn varieties purchased from Stokes Seeds (St. Catherines, Ontario, Canada). Lastly, the lone sorghum genotype was provided by Dr. Om P. Dangi and Dr. K. Anand Kumar of Agriculture

Environmental Renewal Canada Inc. (Delhi, Ontario, Canada).

Table 2.2 General information regarding corn and sorghum genotypes

Genotype Background Additional information

C0258 Grain corn Inbred

C0428 Grain corn Inbred

C0444 Grain corn High sugar content

C0103 Grain corn High sugar content

C0348 Grain corn High sugar content

NSS120 Sweet corn Shrunken-2, yellow

UT128B Sweet corn Shrunken-2, yellow

N111 Sweet sorghum Inbred

2.4 Greenhouse trials

2.4.1 Planting

Prior to planting corn and sorghum, seeds were thoroughly washed with tap water to remove fungicidal residues. The seeds were then surface sterilized with a 1% commercial bleach (6% w/v sodium hypochlorite) solution for 15 min, and rinsed three times with sterilized milli-Q H2O. Seeds were planted into holes 2 cm deep in a 1:3 (v/v) sand:vermiculate mixture in plastic germination trays, with one seed per hole.

In the soil drench inoculation experiments each corn genotype had 20 seeds planted;

10 seeds were for experimental inoculation and 10 seeds were for control inoculation. Two trials of root dip inoculation experiments were conducted. In trial 1, 20 seeds of each corn genotype were planted; 10 seeds were for experimental inoculation, 10 seeds were for control inoculation. In trial 2, 10 seeds of each corn genotype were planted; 6 seeds were for experimental inoculation, 4 seeds were for control inoculation. Sorghum genotype N111 was used as a methodological control in both soil drench and root dip experiments. Twenty seeds were planted for each method of inoculation; 10 seeds for experimental inoculation and 10 seeds for control inoculation. All corn and sorghum genotypes received daily watering and were germinated under standard greenhouse conditions. Seedlings were grown until the 2-3 leaf stage, 14-21 days for corn genotypes and 21-28 days for the sorghum genotype.

2.4.2 Greenhouse inoculation

2.4.2.1 Soil drench

Seedlings were transferred at the 2-3 leaf stage from the germination trays into individual pots containing a 1:3 (v/v) sand:pro mix® (Table 2.3) mixture. Prior to transfer, seedling roots were thoroughly rinsed to ensure the removal of any adhering vermiculite or sand from the germination trays. Once replanted, seedlings were given seven days to reestablish themselves in their new pots prior to inoculation. Bacterial inoculum was prepared from a 48 h grown culture which was collected through centrifugation at 5,000 rpm for 15 min before being re-suspended and diluted to the desired concentration using 0.8%

NaCl. Seedlings were inoculated in triplicate with 15 mL of G. diazotrophicus solution at

~108 CFU/ mL in 0.8% NaCl. Control seedlings received 15 mL of 0.8% NaCl. Bacterial inoculum was confirmed through serial dilution and plating. Soil drench inoculation was accomplished by pouring the inoculum onto the soil in close proximity to the seedling

(Bressan and Borges, 2004).

2.4.2.2 Root dip

When seedlings reached the desired 2-3 leaf growth stage, they were removed from their germination trays and were thoroughly rinsed with tap water to remove any adhering vermiculite and sand. Seedlings then had 10-15% of their roots cut prior to being submerged into the inoculum for 30 min. The inoculum used in the root dip experiments was composed of 30 ml of G. diazotrophicus solution at ~105 CFU/ mL in 0.8% NaCl, and was prepared as

Table 2.3 Pro Mix Components

Premier Pro-Mix BX

Components

 Canadian Sphagnum Peat Moss (80-85%/vol)  Mycorrhizae – endomycorrhizal inoculum (Glomus intraradices)  Perlite – horticultural grade  Vermiculite – horticultural grade  Dolomitic and Calcitic limestone (pH adjuster)  Wetting Agent

described under soil drench methods. Control plants also had 10-15% of their roots cut and were submerged for 30 min in 0.8% NaCl. Following inoculation seedlings were planted into identical potting mix as described with soil drench methods. Bacterial inoculum concentration was confirmed through serial dilution and plating.

2.4.3 Harvesting

Corn and sorghum genotypes were harvested 25 days following inoculation. Once removed from their pots, plants were thoroughly rinsed with tap water to remove adhering sand and soil. Plants were then separated into roots, stems, and leaves and were surface sterilized for 10 min in a 1% commercial bleach (6% v/v sodium hypochlorite) solution and rinsed before being placed into plastic bags. Separated plant tissues were then flash frozen in liquid nitrogen and stored at -80 ºC for later analysis.

2.5 Aseptic trials

2.5.1 Germination

Corn seeds that were planted aseptically were surface sterilized prior to germination.

The sterilization process was as follows: 5 min in 70% (v/v) ethanol, 25 min in a solution containing 50% commercial bleach (6% v/v sodium hypochlorite) with 0.1% tween 20, rinsed five times with sterilized milli-Q H2O. Once surface sterilized, seeds were placed into deep petri dishes (100×25 mm) containing two sheets of 7.5 cm filter paper which were moistened with 3 mL of sterilized milli-Q H2O. Each petri dish contained five seeds and germination took place in the dark at 25 ºC for five to seven days. In total, 50 seeds were

germinated for each corn genotype. Length of germination depended on the developmental stage of the germinating seed.

2.5.2 Planting and inoculating

Seedlings with radicles and coleoptiles measuring at least 10 mm were transferred into Magenta boxes where growth under aseptic conditions continued. Magenta boxes were filled with 50 mL of Murashige and Skoog medium supplemented with Murashige and

Skoog vitamins (2.0 mg glycine, 0.5 mg nicotinic acid, 100 mg myo-inositol, 0.5 mg pyridoxine HCl, 0.1 mg thiamine HCl) and 0.3% (w/v) gelrite. Each Magenta box contained only one seedling. Seedlings were given five days to establish themselves in the Magenta boxes prior to being inoculated. Bacterial inoculum was prepared from a 48 h grown culture which was collected through centrifugation at 5,000 rpm for 15 min before being re- suspended and diluted to 102 CFU/mL using 0.8% NaCl. Each seedling was inoculated with a

1 mL aliquot of the inoculum which was pipetted at the base of the seedling; control seedlings were inoculated with 0.8% NaCl.

2.5.3 Harvesting

Corn seedlings were grown for 20 days following inoculation before being harvested.

Once removed from the Magenta boxes, plants were cleaned in sterile milli-Q H2O to ensure that all adhering growth medium was removed. Plants were surface sterilized in a 1% commercial bleach (6% v/v sodium hypochlorite) solution and rinsed in sterile milli-Q H2O.

Once air dried, plants were placed into plastic bags and flash frozen in liquid nitrogen prior to being stored at -80 ºC for later analysis.

2.6 DNA extraction

DNA was extracted from the corn and sorghum plants. Approximately 1.5 g of tissue was taken from the harvested samples and placed into Bioreba bags to which 1 mL of sterilized milli-Q H2O was added. Samples were then thoroughly ground up using the

Bioreba AG Homex 6 homogenizer. A 100 µL aliquot of the homogenized solution was carefully transferred from the Bioreba bag to an empty 1.5 mL Eppendorf tube ensuring that minimal debris was transferred. Four hundred microliters of extraction buffer (Table 2.4) were subsequently added to the Eppendorf tube and the mixture was vortexed for 5 s. The samples were then centrifuged at 13000 rpm for 5 min and 300 µL of the supernatant were transferred to a clean 1.5 mL Eppendorf tube to which 300 µL of isopropanol was added. The new tube was then shaken and left at room temperature for 30 min before being centrifuged at 13000 rpm for 5 min. Following centrifugation, the isopropanol was discarded and the remaining pellet was washed with 70% EtOH and centrifuged again at 13000 rpm for 1 min.

The EtOH was then discarded and the pellet was left to dry in the tube on the bench for 1-2 h.

When dry, the white DNA along the sides of the tube was re-suspended in 50 µL of sterilized mill-Q H2O by flicking the tube. The H2O which contained DNA was then transferred into a clean 1.5 mL Eppendorf tube. The DNA extract was then held at 4 ºC until analyzed via

PCR.

Table 2.4 Components of the DNA extraction buffer

Extraction Buffer 100 mL

Water 65 mL

200 mM Tris pH 8.0 20 ml 1M

250 mM NaCl 5 ml of 5M

25 mM EDTA 5 ml of 0.5M

0.5% SDS 5 ml of 10%

2.7 Acetylene reduction assay

Nitrogenase activity of the G. diazotrophicus strain Pal5 was tested using the acetylene reduction assay (ARA) method (Hardy et. al., 1968). The Pal5 strain was cultured for 48 h at 28 ºC in LGIP liquid medium supplemented with 10 mM (NH4)2SO4. The bacterial solution was subsequently centrifuged at 5,000 rpm for 15 min before the supernatant was discarded. The bacterial pellet was then twice washed and re-suspended in equal volumes of sterilized milli-Q H2O. Five milliliters of semisolid LGIP (not supplemented with 10 mM (NH4)2SO4) were added to 40 mL vials equipped with a septum cap and inoculated with 100 µL of the re-suspended bacterial culture, 108 CFU/mL confirmed via serial dilution. Bacterial cultures in semisolid LGIP medium were incubated for 48 h at 28 ºC. Following incubation a syringe was used to remove 10% (v/v) of air from the vials and replace it with an equal volume of acetylene (C2H2). Following an additional 48 h of incubation at 28 ºC, the vials were analyzed for the presence of ethylene (C2H4). ARA measurements were conducted using either a Hewlett Packard 5890 Series II gas chromatograph (GC) (Agilent Technologies) with flame ionization detection (FID), or an

Agilent Technologies 7890A/5975C GC-MSD system using mass spectrometry detection.

Either a GS-GASPRO capillary column (30 m × 0.32 mm, Agilent J&W GC columns), or a

CarboxenTM 1006 PLOT fused silica capillary column (30 m × 0.32 mm, Supelco) was used with helium as the carrier gas. Using a gas-tight syringe (Hamilton, Reno, NV, USA) 20 µL was taken from the headspace of each vial and manually injected into the GC. Negative controls were subjected to the same protocols as experimental samples without the addition of bacterial cultures. When using the GS-GASPRO capillary column a splitless injection was made into the inlet set at 250 ºC, the oven was set at 90 ºC, the FID was set at 260 ºC and the

inlet pressure was 20 psi. When using the CarboxenTM 1006 PLOT fused silica capillary column a splitless injection was made into the inlet set at 120 ºC, the inlet pressure was 20 psi, the oven was set at 120 ºC, and the FID temperature was 250 ºC. For MS detection the

MS-source temperature was 230 ºC, the MS-quadrupole was 150 ºC and selected ion monitoring mode was used to monitor m/z 28 with a dwell time of 200 ms. Ethylene standard curves were prepared using various injection volumes of 10, 100, and 1000 ppm ethylene standards in helium (Scotty® Analyzed Gases)

Statistical analysis was conducted with SigmaPlot 12.0 (Systat Software, Inc.

SigmaPlot for Windows). Nitrogenase activity results were compared by one-way analysis of variance. All multiple comparisons were performed by the Tukey’s post-hoc test. All statistical analyses were conducted at P=0.05.

2.8 Sucrose analysis

Sucrose analysis was performed on each of the seven corn genotypes examined in this study. Three plants from each corn genotype were separated into roots, stems, and leaves, and the sucrose concentration of each separate tissue was analyzed by gas chromatography- flame ionization detection (GC-FID). Tissue samples were kept separate from other tissues originating from either the same plant, genotype or tissue type. Frozen samples were freeze- dried for 24-48 h before being individually ground up using a Wiley mill fixed with a No. 20 mesh screen. Two hundred milligram aliquots of the ground up tissue samples were used for sucrose extraction. Each aliquot underwent two rounds of extraction. Samples were placed into 15 mL disposable centrifuge tubes with 5 mL of milli-Q H2O and were first vortexed and then centrifuged at 2500 rpm for 15 min at 24 ºC. Following centrifugation, the supernatant

was transferred into a 15 mL falcon tube for later use. An additional 5 mL of milli-Q H2O were added to the disposable centrifuge tube and the previously described vortexing and centrifugation steps were repeated to extract any remaining sucrose. The two extraction volumes were combined and a 100 µL aliquot was transferred into a 2 mL target DPTM glass reaction vial for centrifugal evaporation using the Savant SVC100H SpeedVac Concentrator.

Following evaporation, dried sucrose extracts underwent methyloximation derivatization for

90 min at 30 ºC with methoxyamine (MOXTM) reagent (2% methoxyamine HCL in pyridine,

ThermoFisher Scientific). Each sample then underwent trimethylsilyl derivatization for 30 min at 37 ºC with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) (Sigma-

Aldrich®). GC-FID analysis was accomplished with the Hewlett Packard 5890 Series II GC equipped with a DB-5 capillary column (30 m × 0.25 mm, 0.25 µm thickness, J&W

Scientific). Two-microliter samples were introduced into the GC-FID via split-less injection at an injection temperature of 280 ºC, and helium was used as the carrier gas with an inlet pressure of 8 psi. The GC-FID column temperature was set to an initial 70 ºC, followed by a ramp at 5 ºC min-1 up to 330 ºC, where it was held for six minutes. The FID temperature was

280 ºC. Known concentrations of sucrose standards were prepared and analyzed to generate a calibration curve for the quantitative calculation of sucrose found within each plant tissue sample. Quantitative calculations were made based on the relative peak area distinguished from the GC-FID produced gas chromatogram.

Statistical analysis was conducted with SigmaPlot 12.0 (Systat Software, Inc.

SigmaPlot for Windows). Sucrose analysis results were compared by one-way analysis of variance. All multiple comparisons were performed by the Tukey’s post-hoc test. All statistical analyses were conducted at P=0.05.

Chapter 3. Results

3.1 Confirmational analysis

3.1.1 Bacterial growth

When G. diazotrophicus strain Pal5 was grown on LGIP plates supplemented with 10 mM NH4(SO4)2, the resulting colonies were smooth with regular edges. Colonies initially appeared semi-transparent but became dark orange in colour following five full days of incubation (Figure 3.1a). When cultured in liquid LGIP supplemented with 10 mM

NH4(SO4)2 growth was observed as a continuous increase in turbidity associated with the increased number of bacteria (Figure 3.1b). On semi-solid LGIP supplemented with 10 mM

NH4(SO4)2, G. diazotrophicus formed an orange pellicle just below the medium’s surface.

The pellicle became darker and thicker throughout incubation (Figure 3.1c). In the absence of

10 mM NH4(SO4)2, the bacterial pellicle was thinner and lighter in colour. When grown in either solid or semi-solid medium, G. diazotrophicus visibly changed the colour of the medium, from light orange to pale yellow.

3.1.2 PCR confirmation

Through PCR analysis it was identified that the Pal5 strain used in this study was G. diazotrophicus. Both sets of primers of the nested PCR were examined independently of each other for their ability to detect the bacterium. Primers GDI25F and GDI923R, used in the

A B

Figure 3.1 Morphology of G. diazotrophicus in different types of LGIP media supplemented with 10 mM NH4(SO4)2 A, G. diazotrophicus plated on solid LGIP medium at 104-106 CFU/mL. B, Control and 48 h incubated G. diazotrophicus in liquid LGIP medium. C, G. diazotrophicus at 108 CFU/mL and control in semi-solid LGIP medium

first round of the nested PCR resulted in an 899 bp product, while primers GDI39F and

GDI916R, used in the second round of the nested PCR, produced a 879 bp product (Figure

3.2). The sensitivity of the nested PCR was tested by examining serial dilutions of the bacterium. Round one of the PCR was capable of detecting bacterial DNA from a dilution factor of 104, while the second round of PCR was able to detect bacterial DNA from a dilution factor of 106 (Figure 3.3). Ubiquitin primers Ub-U29162-F and Ub-U29162-R were tested on corn, sorghum, and bacterial samples but only produced the expected 218 bp product with the corn and sorghum plant samples (Figure 3.4).

3.1.3 Nitrogenase activity confirmation

An acetylene reduction assay was performed on G. diazotrophicus at 104 and 108

CFU/mL grown in different media (semi-solid LGIP supplemented and not supplemented with 10 mM NH4(SO4)2) and incubated for different durations (8, 24, and 48 h). Results were converted to nM of ethylene through the use of ethylene standard curves (Figure 3.5).

Following the ARA, it was determined that with an 8 h incubation period neither the bacterium at 104 nor 108 CFU/mL were capable of producing a detectable amount of ethylene when grown in LGIP supplemented with 10 mM NH4(SO4)2. Additionally, while the bacterium grown in nitrogen-free LGIP produced a detectable amount of ethylene, no significant difference was observed between the bacterium at 104 and 108 CFU/mL and the amount of ethylene was not significantly different from the 10 mM NH4(SO4)2 supplemented trials. In the 24 h incubation trials, no ethylene was detected from the bacterium grown in 10 mM NH4(SO4)2 supplemented LGIP. However, cultures grown in nitrogen-free LGIP

Figure 3.2 Nested PCR amplification of G. diazotrophicus Pal5 strain Lane 1 – 100 bp ladder, lanes 2-3 – Round 1 Pal5 102 dilution, lanes 4-5 – Round 2 Pal5 102 dilution, lane 6 – 100 bp ladder

A B

Figure 3.3 Nested PCR sensitivity for Pal5 strain A, Round 1 of nested PCR; B, Round 2 of nested PCR Lane 1 – 100 bp ladder, lane 2 – undiluted colony, lane 3 – 101 dilution, lane 4 – 102 dilution, lane 5 – 103 dilution, lane 6 – 104 dilution, lane 7 – 105 dilution, lane 8 – 106 dilution, lane 9 – 107 dilution, lane 10 – 100 bp ladder

Figure 3.4 PCR amplification of ubiquitin Lane 1 – 100 bp ladder, lane 2-3 – Z. mays tissue sample, lanes 4-5 – S. bicolor tissue sample, lanes 6-7 – Pal5 102 dilution, lane 8 – 100 bp ladder

Figure 3.5 Ethylene standard Curves Standard curves obtained using a 10 ppm (A), 100 ppm (B), and 1000 ppm (C) C2H4 in helium standard for the quantification of C2H4 from ARA

produced significantly more ethylene compared to the bacterium grown in 10 mM NH4(SO4)2 supplemented LGIP (P<0.001). In the 24 h incubated nitrogen-free experiments, a significant difference in ethylene production was observed between the 108 CFU/mL trials when compared to the 104 CFU/mL trials (P=0.011). In the 48 h incubation trials, the bacterium grown in LGIP medium supplemented with 10 mM NH4(SO4)2 produced a detectable amount of ethylene; no significant difference was observed between 104 and 108 CFU/mL samples.

Bacteria grown in nitrogen-free LGIP produced a significantly higher amount of ethylene in comparison to those grown with nitrogen supplementation (P<0.001). Within the samples grown in the nitrogen-free environment, the 108 CFU/mL sample produced a significantly larger amount of ethylene compared to the 104 CFU/mL sample (P<0.001). No significant difference in ethylene production was observed in the nitrogen supplemented trials across all three incubation periods for both the 104 and 108 CFU/mL samples. Lastly, within the bacterium grown in a nitrogen-free environment, a significant difference in ethylene production by the bacterium at 104 CFU/mL was observed over the three different incubation periods (P<0.002). The same was observed for the bacterium grown at 108 CFU/mL

(P<0.001) (Table 3.1).

3.2 Green house trials

3.2.1 Soil drench trials

Following germination, inoculation, and maturation plants in the soil drench trials were harvested and separated into roots, stems, and leaves to be examined through nested

PCR for the presence of G. diazotrophicus. PCR confirmation of ubiquitin was conducted to

Table 3.1 Nitrogenase activity of G. diazotrophicus measured by ARA

Ethylene produced (nM) by Gluconacetobacter diazotrophicus over specified incubation periods

Colony forming units and 8 hours 24 hours 48 hours nitrogen source

4 A A A 10 CFU/mL 10 mM NH4(SO4)2 0 0 4.89 ± 0.17

8 A A A 10 CFU/ mL 10 mM NH4(SO4)2 0 0 11.1 ± 2.57

104 CFU/mL Nitrogen-free 50.9 ± 2.26A 464 ± 55.1BC 618 ± 55.5CD

108 CFU/mL Nitrogen-free 175 ± 19.0AB 892 ± 68.6D 1736 ± 333E

Results are mean ± SD for three replicates of each treatment. Significant differences in nitrogenase activity were visible among the incubation periods and different bacterial CFU’s coupled with nitrogen sources (F=84.035, DF=11, P<0.001). Different letters denote significantly different nitrogenase activity following a multiple comparisons Tukey’s test (P<0.05).

confirm that the DNA extraction process was successful. G. diazotrophicus DNA was not detected in any of the inoculated or control plants analyzed. Ubiquitin was detected in all inoculated and control plants analyzed (Appendix A.2-A.7).

Sorghum genotype N111 was used as a methodological control. Following round one of PCR analysis G. diazotrophicus was detected in the roots of 30% of plants and in the stems of 20% of plants. Following round 2 of nested PCR the bacterium was detected in the roots of 40% of plants and in the stems of 20% of plants (Appendix A.8-A.10). Equation 3.1 was used to calculate the bacterium’s colonization efficiency.

Equation 3.1 Colonization efficiency

PCR positive plants/ tissues Colonization efficiency = × 100 Inoculated plants/ tissues

Using the soil drench method of inoculation, G. diazotrophicus had a colonization efficiency of 40% within the root tissue, and 20% within the stem tissue of sorghum genotype N111. G. diazotrophicus was not detected in any of the experimental leaf samples and in any of the control plants. Ubiquitin was detected in all experimental and control plants analyzed (Tables 3.2 and 3.3). Equation 3.2 was used to calculate a method’s inoculation efficiency. Overall the soil drench method of inoculation had an inoculation efficiency of 0% within corn and 40% within sorghum.

Equation 3.2 Inoculation efficiency

PCR positive plants Inoculation efficiency = × 100 Inoculated plants

Table 3.2 Analysis of bacterial presence in soil drench inoculated samples of greenhouse sorghum via PCR Genotype – N111

Tissue PCR Round 1 PCR Round 2 Ubiquitin

Root 3 (10) 4 (10) 10 (10)

Stem 2 (10) 2 (10) 10 (10)

Leaves 0 (10) 0 (10) 10 (10)

Note: Numbers in brackets signify total number of samples analysed

Table 3.3 Analysis of bacterial presence in soil drench inoculated control samples of greenhouse sorghum via PCR Genotype – N111

Tissue PCR Round 1 PCR Round 2 Ubiquitin

Root 0 (4) 0 (4) 4 (4)

Stem 0 (4) 0 (4) 4 (4)

Leaves 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

3.2.2 Root dip trials

Two trials of root dip inoculation experiments were conducted. In trial 1, following nested PCR analysis, G. diazotrophicus DNA was not detected in any of the experimental or control plants analyzed. Ubiquitin was detected in all experimental and control plants analyzed (Appendix A.11-A.16). In trial 2, following nested PCR analysis, G. diazotrophicus

DNA was not detected in any of the experimental or control plants analyzed. Ubiquitin was detected in all experimental and control plants analyzed (Appendix A.17-A.22).

Sorghum genotype N111 was used as a methodological control. PCR analysis of experimental sorghum plants revealed that G. diazotrophicus DNA was detected in 60% of root samples, 40% of stem samples, and 30% of leaf samples following round 1 of the nested

PCR. Round 2 of nested PCR revealed that G. diazotrophicus DNA was detected in a total of

80% of root samples, 60% of stem samples, and 40% of leaf samples (Appendix A.23-A.25).

Using the root dip method of inoculation, G. diazotrophicus had a colonization efficiency of

80% within the root tissue, 60% within the stem tissue, and 40% within the leaf tissue of sorghum genotype N111. G. diazotrophicus DNA was not detected in any of the control plants analyzed. Ubiquitin was detected in all experimental and control plants analyzed

(Tables 3.4 and 3.5). Overall, the root dip method of inoculation had an inoculation efficiency of 0% within corn and 80% within sorghum.

3.3. Aseptic trials

A subset of the harvested plants was analyzed for the colonization of G. diazotrophicus. In total, 10 experimental plants and 4 control plants were analyzed within

Table 3.4 Analysis of bacterial presence in root dip inoculated samples of greenhouse sorghum via PCR Genotype – N111

Tissue PCR Round 1 PCR Round 2 Ubiquitin

Root 6 (10) 8 (10) 10 (10)

Stem 4 (10) 6 (10) 10 (10)

Leaves 3 (10) 4 (10) 10 (10)

Note: Numbers in brackets signify total number of samples analysed

Table 3.5 Analysis of bacterial presence in root dip inoculated control samples of greenhouse sorghum via PCR Genotype – N111

Tissue PCR Round 1 PCR Round 2 Ubiquitin

Root 0 (4) 0 (4) 4 (4)

Stem 0 (4) 0 (4) 4 (4)

Leaves 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

each genotype, with the exception of genotype UT128B in which only 9 experimental plants were analyzed. DNA analysis of the seven corn genotypes led to the conclusion that colonization efficiency of G. diazotrophicus using the aseptic method of inoculation was

100% in C0444 plants, 90% in C0348 plants, 90% in C0103 plants, 100% in C0428 plants,

90% in C0258 plants, 100% in NSS120 plants, and 100% in UT128B plants (A 3.6-3.12). G. diazotrophicus was not detected in any of the control plants. Ubiquitin was detected in all experimental and control plants analyzed (Tables 3.6-3.7). Overall, the aseptic method of inoculation had an inoculation efficiency of 93% within corn.

3.4 Acetylene reduction assay analysis

Acetylene reduction assays were performed on experimental plants from the aseptic trials that were not used in DNA analysis. As inoculation efficiency under aseptic conditions was 93%, it was presumed that most of the plants analyzed had been successfully colonized.

Additionally, tissue from PCR positive sorghum samples was also analyzed. Ethylene was not detected in any of the corn plants or sorghum tissue samples analyzed (Tables 3.8-3.10).

3.5 Sucrose analysis

Sucrose levels in the root, stem, and leaf tissues were analyzed across all seven corn genotypes. In the grain corn genotypes sucrose levels within the roots, stems, and leaves ranged from 0.13-25.16 mg/g dry weight, 3.58-4.96 mg/g dry weight, and 0.004-0.04 mg/g dry weight respectively. In the high sucrose grain corn genotypes sucrose levels

Table 3.6 Analysis of bacterial presence in aseptically inoculated corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 3 (10) 10 (10) 10 (10)

C0348 3 (10) 9 (10) 10 (10)

C0103 3 (10) 9 (10) 10 (10)

C0428 7 (10) 10 (10) 10 (10)

C0258 3 (10) 9 (10) 10 (10)

NSS1120 3 (10) 10 (10) 10 (10)

UT128B 1 (9) 9 (9) 9 (9)

Note: Numbers in brackets signify total number of samples analysed

Table 3.7 Analysis of bacterial presence in aseptically inoculated control corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (4) 0 (4) 4 (4)

C0348 0 (4) 0 (4) 4 (4)

C0103 0 (4) 0 (4) 4 (4)

C0428 0 (4) 0 (4) 4 (4)

C0258 0 (4) 0 (4) 4 (4)

NSS1120 0 (4) 0 (4) 4 (4)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

Table 3.8 ARA analysis of aseptically inoculated corn

Genotype Ethylene detected

C0103 0 (3)

C0348 0 (3)

C0444 0 (3)

C0425 0 (3)

C0258 0 (1)

NSS120 0 (3)

UT128B 0 (3)

Note: Numbers in brackets signify total number of samples analysed

Table 3.9 ARA analysis of soil drench inoculated sorghum genotype N111

Tissue Ethylene detected

Root 0 (4)

Stem 0 (2)

Note: Numbers in brackets signify total number of samples analysed

Table 3.10 ARA analysis of root dip inoculated sorghum genotype N111

Tissue Ethylene detected

Root 0 (8)

Stem 0 (6)

Leaf 0 (4)

Note: Numbers in brackets signify total number of samples analysed

within the roots, stems, and leaves ranged from 30.3-81.2 mg/g dry weight, 43.6-210 mg/g dry weight, and 0.06-21.4 mg/g dry weight, respectively. Lastly, sucrose levels within the roots, stems, and leaves of sweet corn genotypes ranged from 115-128 mg/g dry weight, 221-

292 mg/g dry weight, and 14.5-22.4 mg/g dry weight, respectively. Sucrose concentrations were significantly different between all tissue samples amongst the grain corn and sweet corn genotypes (P<0.05) (Figures 3.13-3.15). Results varied with the new high sucrose grain corn genotypes. C0103 did not contain a significantly higher sucrose level than both of the grain corn genotypes in any of the tissues analyzed. C0348 contained significantly higher sucrose levels within its stem compared to regular grain corn (P<0.001) but was not significantly different when compared to sweet corn. However, within the leaf C0348 contained significantly less sucrose in comparison to sweet corn (P<0.001), but was not significantly different in comparison to grain corn. The stem of C0348 contained sucrose levels that were not significantly different than either the grain corn or sweet corn. Lastly, C0444 contained stem and leaf sucrose levels significantly higher than those of the grain corn genotypes

(P<0.001), but not significantly different from the sweet corn genotypes; C0444 root sucrose levels were not significantly different than either the grain corn or sweet corn.

A B

A B C

C C

Figure 3.6 Sucrose concentrations in the roots of seven corn genotypes Results are mean ± SE for three replicates of each genotype. Significant differences in sucrose concentrations were observed among the various corn genotypes. (F=8.309, DF=6, P<0.001). Different letters denote significantly different sucrose concentrations following a multiple comparisons Tukey’s test (P<0.05).

A B B A B

Figure 3.8 Sucrose concentrations in the stems of seven corn genotypes Results are mean ± SE for three replicates of each genotype. Significant differences in sucrose concentrations were observed among the various corn genotypes. (F=41.53, DF=6, P<0.001). Different letters denote significantly different sucrose concentrations following a multiple comparisons Tukey’s test (P<0.05).

A A

B B B

Figure 3.7 Sucrose concentrations in the leaves of seven corn genotypes Results are mean ± SE for three replicates of each genotype. Significant differences in sucrose concentrations were observed among the various corn genotypes. (F=36.399, DF=6, P<0.001). Different letters denote significantly different sucrose concentrations following a multiple comparisons Tukey’s test (P<0.05).

Chapter 4. Discussion and Conclusion

4.1 Laboratory grown cultures of G. diazotrophicus

Gluconacetobacter diazotrophicus strain Pal5 was successfully cultured under laboratory conditions. LGIP medium, as described by Cavalcante and Dobereiner (1988), was used to culture the bacterium, because its high sucrose concentration best replicated the levels found within its natural host, sugarcane. When grown on solid LGIP medium supplemented with 10 mM NH4(SO4)2 as its nitrogen source, G. diazotrophicus initially formed small round white colonies which gradually turned yellow and lastly a dark shade of orange, as described by Cavalcante and Dobereiner (1988). The colour change is due to the uptake of the bromothymol blue from within the LGIP medium, which in turn changed the colour of the medium from light orange to pale yellow; the same was observed in the semi- solid medium. When cultured in semi-solid medium, the bacterium forms a pellicle as described by Cavalcante and Dobereiner (1988) just beneath the medium’s surface. The pellicle’s darker and thicker appearance in the presence of supplemented nitrogen illustrated that the bacterium’s growth was nitrogen-dependent. These key characteristics of growth under laboratory conditions by the bacterium fit the descriptions detailed in past studies and confirm based on phenotype that the bacterium used in this study was G. diazotrophicus

(Cavalcante and Dobereiner, 1988; Gillis et al., 1988).

4.2 PCR verification

The identity of Gluconacetobacter diazotrophicus was verified through PCR analysis prior to start of this study. Primers designed by Franke-Whittle et al. (2005) and Tian et al.

(2009) were used individually to identify the bacterial strain as G. diazotrophicus. Both sets of primers were based on the 16S rDNA of the bacterium and were used in succession, because the PCR primers designed by Tian et al (2009) were created to produce a product that could be amplified by the primers of Franke-Whittle et al. (2005). The nested PCR process was very useful in detecting the bacterium in low concentrations, as discussed by

Tian et al. (2009). With the use of a nested PCR the sensitivity for detection of G. diazotrophicus was greatly increased, because the nested PCR was capable of identifying the bacterium from samples containing a dilution factor of 106, while the single primer set was only capable of detecting the bacterium from samples containing a dilution factor of 104.

This increased sensitivity was imperative to ensure that bacterial colonization of plant tissues was not missed due to low colonization numbers.

To ensure that the DNA extraction process was successful in situations in which no colonization occurred, the identification of a ubiquitous corn protein was necessary. Corn ubiquitin was selected, as the protein is ubiquitous and found throughout the plant’s tissues.

Primers designed by Sorgona et al. (2011) were used, and due to genetic similarities between the monocots corn and sorghum, the primers were also capable of detecting sorghum ubiquitin. Analyses of the ubiquitin primers with G. diazotrophicus confirmed that the PCR was not capable of amplifying anything from the bacterium’s DNA.

4.3 Nitrogenase activity of G. diazotrophicus

Prior to the start of this study the nitrogenase activity of G. diazotrophicus was confirmed through an acetylene reduction assay. Nitrogenase activity was measured through its ability to convert acetylene into ethylene, a process that mirrors the enzyme’s dinitrogen

fixing capabilities (Dilworth, 1966). Although this method does not provide an accurate estimate for the actual amount of N2 fixation, it is a fast and inexpensive procedure capable of determining the activity of nitrogenase (Madigan and Martinko, 2006). N2 fixation can be measured accurately with the use of 15N isotopes (Lima et al., 1987; Boddey et al., 2001).

Several different factors influenced nitrogenase activity of G. diazotrophicus in this study.

One of the main factors was the addition of 10 mM NH4(SO4)2 as the nitrogen source.

Although the bacterium visually produced a thicker and darker pellicle, indicating greater colony size, its nitrogenase activity, measured by its production of nM ethylene, was significantly less when compared to trials not supplemented with a nitrogen source. The G. diazotrophicus nitrogenase is partially inhibited by ammonium at low concentrations

(Stephan et al., 1991). However, at the 10 mM concentration, ammonium completely inhibited the nitrogenase, as was observed in the 8 and 24 h trials. Nitrogenase activity was detected in the 48 h trial, indicating that the bacterium used up all the available ammonium from the medium and was required to fix nitrogen, as expected due to the fact that its growth is nitrogen-dependent (Cavalcante and Dobereiner 1988). In addition to the inhibitory actions of ammonium, the bacterium’s own product of N2 fixation, ammonia, is capable of inhibiting nitrogenase (Madigan and Martinko, 2006). As the bacterium undergoes N2 fixation and produces ammonia it is immediately used in biosynthesis. However, as excess ammonia begins to accumulate nitrogenase is switched-off through a feed-back inhibition process, which ensures that available energy is diverted from the high-energy-demanding process of

N2 fixation. Ammonia inhibits nitrogenase by reversibly binding to the nitrogenase’s MoFe protein, effectively blocking dinitrogen from the binding site (Madigan and Martinko, 2006).

The duration for which the bacterium was incubated was another factor that influenced the

amount of ethylene that was produced. Specifically, within the nitrogen-free trials a significantly larger amount of ethylene was produced when the bacterium was incubated for longer periods of time with the acetylene. Incubation periods longer than 48 h were not analysed in an attempt to avoid bacterial overgrowth. Based on these results, the optimal incubation period for colonized plant samples with acetylene should be 48 h. Lastly, CFU/ mL affected ethylene production, higher CFU numbers produced significantly larger amounts of ethylene, indicating that ethylene production would still be detected within samples containing low CFU numbers.

4.4 Gluconacetobacter diazotrophicus colonization under greenhouse conditions

As an obligate endophyte, G. diazotrophicus is unable to survive outside of a host plant for long periods of time. Baldani et al. (1997) observed that two days following inoculation into unsterilized soil the bacterium was undetectable. This indicates that when inoculated via soil drench, the bacterium has 48 h to enter the host before dying off. Both the soil drench and the root dip methods of inoculation are capable of introducing endophytic bacteria into corn; the root dip method of inoculation was found to be more efficient than the soil drench method (Bressan and Borges, 2004). Specifically with G. diazotrophicus, Tian et al. (2009) have shown that inoculation of this bacterium into several of the grain and sweet corn genotypes used in this study is possible through the root dip method of inoculation.

While results from the current study show that under greenhouse conditions G. diazotrophicus is unable to colonize corn with both soil drench and root dip methods of inoculation, the DNA extraction method used in the current study was different from that used by Tian et al.’s (2009). When the DNA extraction process used by Tian et al. (2009)

was performed on inoculated and un-inoculated samples, ubiquitin and bacterial DNA were never detected through PCR analysis, necessitating changes to the DNA extraction process used in this study. However, as both methods of inoculation resulted in colonization of the sorghum genotype, an underlying problem regarding the interactions between the bacterium and corn genotypes could be responsible. As the corn genotypes were only inoculated after reaching the 2-3 leaf stage, ample time was available for other endophytic bacteria to establish themselves within the corn plants, and thus potentially inhibit both colonization and establishment of G. diazotrophicus. Future studies should use a bacterial control alongside G. diazotrophicus to ensure that colonization is still attainable following possible establishment by other endophytic bacteria; Bacillus spp. as used by Bressan and Borges (2004) could be a suitable bacterial control.

The results obtained in this study for the colonization of sorghum by the root dip method of inoculation, were comparable to those observed in Vanessa Yoon’s study

(unpublished data). Differences between the inoculation efficiencies of soil drench and root dip methods within sorghum were similar to those observed by Bressan and Borges with corn

(2004). PCR analysis of the colonized sorghum plants, both with the soil drench and root dip trials, showed that the primary area of colonization occurred through the roots and systemically spread to the other tissues. There were no incidences where G. diazotrophicus was present in the stem tissues which originated from the same plant containing root tissues in which no bacteria was detected; there were also no incidences of positive leaf tissue originating from plants containing negative root and stem tissues. Regarding the root dip trials, entrance through the root into the plants most likely occurred at the sites which were intentionally pruned in order to create an open wound for the bacterium to use. Within the

soil drench trials, the bacterium would have gained entry into the host’s root system at sites of lateral root emergence, between the cells of the root meristem, or through naturally occurring wounds due to root growth (James et al., 2001; Bressan and Borges, 2004).

Future studies into the interaction between G. diazotrophicus and corn should focus on other methods of inoculation. These include seed coating, a method which, like the soil drench method, has the ability to be implemented into a large scale farming operation. The seed coating method has had positive results in interactions between G. diazotrophicus and corn, sorghum and wheat (Riggs et al., 2001; Luna et al., 2010).

4.5 Gluconacetobacter diazotrophicus colonization under aseptic conditions

Although colonization of corn plants was unsuccessful under greenhouse conditions, the aseptic method of inoculation proved to be very effective. Each of the seven corn genotypes was successfully colonized and the colonization efficiency was at least 90%.

These results support Tian et al.’s (2009) findings that under aseptic conditions the colonization of corn by G. diazotrophicus is achievable. The bacterium did not prefer colonization of one genotype over another, as each genotype had similar colonization efficiencies between 90-100%. While colonization efficiencies were near 100%, the majority of PCR detection occurred only during the second round of PCR analysis, indicating that although colonization occurred, bacterial numbers were very low. While G. diazotrophicus is capable of colonizing these corn genotypes with the aseptic method of inoculation, its inability to do so via soil drench and root dip must be due to the conditions within the greenhouse, as both the soil drench and root dip methods alone both proved successful at introducing the bacterium into sorghum. When grown under aseptic conditions, the corn

seedlings were not exposed to any potential endophytic bacteria, outside of what may already be present and established within the seed (Johnston-Monje and Raizada, 2011). Without the presence of any additional endophytic bacteria, G. diazotrophicus was capable of successfully establishing itself within the corn plant. Additionally, as growth under aseptic conditions occurred on Murashige and Skoog medium, the chance for bacterial survival outside of a host for over 48 h increases greatly. Unlike the Murashige and Skoog medium, unsterilized soil might not contain the nutrients that G. diazotrophicus requires to survive

(Baldani et al., 1997). Furthermore, the bacterial inoculant has the capability of enveloping the host’s roots which have burrowed through the medium and remain there until a suitable method of entry into the plant is present. Therefore, unlike the soil drench and root dip methods, the aseptic method of inoculation provides the bacterium with a hospitable environment in which survival beyond 48 h is possible, longer opportunities to successfully inoculate, and an environment free of potentially inhibiting endophytic bacteria.

4.6 Nitrogenase activity within colonized corn

Aseptically grown corn plants that were not pulverized in the DNA extraction process were used for the acetylene reduction assay analysis. As colonization of aseptically grown plants was at least 90%, it was presumed that the majority of the plants analyzed were colonized by G. diazotrophicus. In addition to corn plants, remaining sorghum tissue samples which had tested positive for G. diazotrophicus colonization were also analysed. Plant and tissue samples were incubated for 48 h with acetylene, as earlier ARA analysis showed that to be an optimal incubation period; longer incubation periods resulted in fungal contamination. Following ARA analysis it was confirmed that ethylene was not detected in

any of the corn plant and sorghum tissue samples. Low bacterial numbers could be one possibility for the lack of nitrogenase activity, as initially suggested by Tian et al. (2009). As mentioned earlier, in a large number of samples, G. diazotrophicus was only detected in the second round of the nested PCR, indicating that there must have been low bacterial numbers in the samples. If nitrogenase is not active due to low bacterial numbers, quorum sensing could be responsible. A complete genome sequence of the Pal5 strain of G. diazotrophicus revealed three genes associated with quorum sensing (Bertalan et al., 2009). Nitrogen fixation regulated by quorum sensing has been previously identified within Rhizobium etli

(Daniels et al. 2002). The lux R/I quorum sensing system within G. diazotrophicus should be a future research target in attempts to establish nitrogenase activity within corn (Reading and

Sperandio, 2006; Bertalan et al., 2009).

4.7 Effect of sucrose on G. diazotrophicus colonization

Sucrose content is very important for the growth of G. diazotrophicus (Cavalcante and Dobereiner, 1988). When the bacterium is cultured in laboratory settings, LGIP medium containing 10% sucrose is used. The natural host plant of the bacterium, sugarcane, is capable of containing sucrose levels of 480 mg/g dry weight with a theoretical maximum of

620 mg/g dry weight (Muchow et al., 1996; Sachdeva et al., 2011). In corn, sucrose is formed in the leaves and is then transferred via the phloem into heterotrophic areas of the plant

(Bruneau et al., 1991; Zhou et al., 1997). My study revealed that on average the stems of the corn plants contained the highest levels of sucrose, the roots contained the second highest levels of sucrose, and the leaves contained the least amount of sucrose. Across the different types of corn plants, the sweet corn varieties had the highest levels of sucrose, especially in

the stems where levels averaged 291 mg/g dry weight in the UT128B genotype. Additionally, two of the newly bred high sucrose grain corn genotypes, C0444 and C0348, showed a significantly higher level of sucrose within their stems compared to the other grain corn genotypes, an important fact considering the majority of corn grown is grain corn. Regarding the sorghum variety used in the greenhouse trials, analysis completed by Vanessa Yoon

(unpublished data) indicate that sucrose levels within sorghum genotype N111 are not significantly higher than any of the sweet corn genotypes used in this study, meaning that the sucrose concentrations alone did not influence G. diazotrophicus colonization of sorghum genotype N111. In the aseptic trials, G. diazotrophicus colonization efficiency was at or above 90% in each corn genotype, indicating that sucrose levels within the plants did not affect colonization efficiency, because significant differences in sucrose levels were present amongst the different genotypes. While my study did not support a correlation between sucrose content and colonization efficiency, more research needs to be conducted with other methods of inoculation. Sucrose will always be an important factor to the colonization success of G. diazotrophicus, because the majority of the bacterium’s natural host plants contain high levels of sucrose (Muthukumarasamy et al., 2002). Additionally, when looking specifically at the process of nitrogen fixation, sucrose levels play an invaluable role, by providing the bacterium with a sufficient source of energy for the process of nitrogen fixation

(Galar and Boiardi, 1995).

4.8 Conclusions

The importance of G. diazotrphicus is unquestionable. This bacterium has been credited with being one of the major factors behind the success of Brazil’s bioethanol fuel program (Medeiros et al., 2006). It supplies sugarcane crops with both a significant amount of fixed nitrogen and plant growth hormones (Fuentes-Ramirez, 1993; Fisher and Newton,

2005). If these properties could be carried over into corn with successful colonization, the resulting impact could be beneficial to both farmers and the environment. Reducing the amount of nitrogen fertilizers applied to corn fields would result in a greater profit margin for farmers, and would result in less damage to the surrounding environment (Duffy, 2009;

Robertson and Vitousek, 2009). The plant hormones produced by the bacterium are capable of increasing crop yields, once again benefiting the farmer (Riggs et al., 2001; Suman et al.,

2005). My study found that with the corn genotypes used, colonization under greenhouse conditions via the soil drench and root dip methods of inoculation was not detected. As the aseptically inoculated plants were successfully colonized, colonization of G. diazotrophicus under greenhouse conditions may not have occurred because of other endophytic bacteria already established within the corn plants, as suggested by the apoplastic sucrose hypothesis

(Riggs et al., 2001) Furthermore, while my study found no correlation between colonization efficiency and sucrose content within the examined corn genotypes, sucrose content remains a critical factor in the successful endophytic establishment of this bacterium. The high rate of colonization due to favourable conditions using the aseptic method of inoculation could have lowered the impact of sucrose content on the colonization of G. diazotrophicus. Therefore, seed inoculation experiments under greenhouse conditions should be attempted with this bacterium and the corn genotypes used in this study.

No nitrogenase activity was detected in this study in plants and tissues colonized by

G. diazotrophicus. If the cause of the enzyme’s inactivity was a result of insufficient quorum sensing signals resulting from low bacterial numbers, potential future experiments should investigate this pathway and pursue means of overriding it (Bertalan et al., 2009). The potential benefits from the successful introduction of G. diazotrophicus into corn are too great to not continue research into this field. With the recent sequencing of the G. diazotrophicus genome, many new directions for future research exist in attaining successful colonization and nitrogen fixation within corn (Bertalan et al., 2009).

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Appendix

A.1 LGIP medium

Components Amount for 1 Liter

K2HPO4 0.2 g

KH2PO4 0.6 g

MgSO4∙7H2O 0.2 g

CaCl2∙2H2O 0.02 g

Na2MoO4∙2H2O 0.002 g

FeCl3∙6H2O 0.01 g

Bromothymol blue in 0.2M KOH 0.025 g

Sucrose 100 g

Yeast extract 0.025 g

Agar (semisolid medium) 4 g

Agar (solid medium) 15 g pH adjusted to 5.5 with 1% acetic acid solution

(Cavalcante and Dobereiner, 1988)

A.2 Analysis of bacterial presence in soil drench inoculated root samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (9) 0 (9) 9 (9)

C0348 0 (9) 0 (9) 9 (9)

C0103 0 (8) 0 (8) 8 (8)

C0428 0 (8) 0 (8) 8 (8)

C0258 0 (9) 0 (9) 9 (9)

NSS1120 0 (9) 0 (9) 9 (9)

UT128B 0 (7) 0 (7) 7 (7)

Note: Numbers in brackets signify total number of samples analysed

A.3 Analysis of bacterial presence in soil drench inoculated stem samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (9) 0 (9) 9 (9)

C0348 0 (9) 0 (9) 9 (9)

C0103 0 (8) 0 (8) 8 (8)

C0428 0 (8) 0 (8) 8 (8)

C0258 0 (9) 0 (9) 9 (9)

NSS1120 0 (9) 0 (9) 9 (9)

UT128B 0 (7) 0 (7) 7 (7)

Note: Numbers in brackets signify total number of samples analysed

A.4 Analysis of bacterial presence in soil drench inoculated leaf samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (9) 0 (9) 9 (9)

C0348 0 (9) 0 (9) 9 (9)

C0103 0 (8) 0 (8) 8 (8)

C0428 0 (8) 0 (8) 8 (8)

C0258 0 (9) 0 (9) 9 (9)

NSS1120 0 (9) 0 (9) 9 (9)

UT128B 0 (7) 0 (7) 7 (7)

Note: Numbers in brackets signify total number of samples analysed

A.5 Analysis of bacterial presence in soil drench inoculated control root samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (10) 0 (10) 10 (10)

C0348 0 (8) 0 (8) 8 (8)

C0103 0 (9) 0 (9) 9 (9)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (8) 0 (8) 8 (8)

NSS1120 0 (9) 0 (9) 9 (9)

UT128B 0 (6) 0 (6) 6 (6)

Note: Numbers in brackets signify total number of samples analysed

A.6 Analysis of bacterial presence in soil drench inoculated control stem samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (10) 0 (10) 10 (10)

C0348 0 (8) 0 (8) 8 (8)

C0103 0 (9) 0 (9) 9 (9)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (8) 0 (8) 8 (8)

NSS1120 0 (9) 0 (9) 9 (9)

UT128B 0 (6) 0 (6) 6 (6)

Note: Numbers in brackets signify total number of samples analysed

A.7 Analysis of bacterial presence in soil drench inoculated control leaf samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (10) 0 (10) 10 (10)

C0348 0 (8) 0 (8) 8 (8)

C0103 0 (9) 0 (9) 9 (9)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (8) 0 (8) 8 (8)

NSS1120 0 (9) 0 (9) 9 (9)

UT128B 0 (6) 0 (6) 6 (6)

Note: Numbers in brackets signify total number of samples analysed

Pal5 Round 1

Pal5

Round 2

Ubiquitin

A.8 Root tissue PCR analysis of soil drench inoculated sorghum genotype N111 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – positive control (Pal5 102 dilution), lanes 14-15 – negative control, lane 16 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – negative control, lane 14 – 100 bp ladder.

Pal5

Round 1

Pal5

Round 2

Ubiquitin

A.9 Stem tissue PCR analysis of soil drench inoculated sorghum genotype N111 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – positive control (Pal5 102 dilution), lanes 14-15 – negative control, lane 16 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – negative control, lane 14 – 100 bp ladder.

Pal5 Round 1

Pal5

Round 2

Ubiquitin

A.10 Leaf tissue PCR analysis of soil drench inoculated sorghum genotype N111 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – positive control (Pal5 102 dilution), lanes 14-15 – negative control, lane 16 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – negative control, lane 14 – 100 bp ladder.

A.11 Trial 1 analysis of bacterial presence in root dip inoculated root samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (9) 0 (9) 9 (9)

C0348 0 (10) 0 (10) 10 (10)

C0103 0 (8) 0 (8) 8 (8)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (10) 0 (10) 10 (10)

NSS1120 0 (10) 0 (10) 10 (10)

UT128B 0 (7) 0 (7) 7 (7)

Note: Numbers in brackets signify total number of samples analysed

A.12 Trial 1 analysis of bacterial presence in root dip inoculated stem samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (9) 0 (9) 9 (9)

C0348 0 (10) 0 (10) 10 (10)

C0103 0 (8) 0 (8) 8 (8)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (10) 0 (10) 10 (10)

NSS1120 0 (10) 0 (10) 10 (10)

UT128B 0 (7) 0 (7) 7 (7)

Note: Numbers in brackets signify total number of samples analysed

A.13 Trial 1 analysis of bacterial presence in root dip inoculated leaf samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (9) 0 (9) 9 (9)

C0348 0 (10) 0 (10) 10 (10)

C0103 0 (8) 0 (8) 8 (8)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (10) 0 (10) 10 (10)

NSS1120 0 (10) 0 (10) 10 (10)

UT128B 0 (7) 0 (7) 7 (7)

Note: Numbers in brackets signify total number of samples analysed

A.14 Trial 1 analysis of bacterial presence in root dip inoculated control root samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (10) 0 (10) 10 (10)

C0348 0 (10) 0 (10) 10 (10)

C0103 0 (9) 0 (9) 9 (9)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (10) 0 (10) 10 (10)

NSS1120 0 (10) 0 (10) 10 (10)

UT128B 0 (9) 0 (9) 9 (9)

Note: Numbers in brackets signify total number of samples analysed

A.15 Trial 1 analysis of bacterial presence in root dip inoculated control stem samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (10) 0 (10) 10 (10)

C0348 0 (10) 0 (10) 10 (10)

C0103 0 (9) 0 (9) 9 (9)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (10) 0 (10) 10 (10)

NSS1120 0 (10) 0 (10) 10 (10)

UT128B 0 (9) 0 (9) 9 (9)

Note: Numbers in brackets signify total number of samples analysed

A.16 Trial 1 analysis of bacterial presence in root dip inoculated control leaf samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (10) 0 (10) 10 (10)

C0348 0 (10) 0 (10) 10 (10)

C0103 0 (9) 0 (9) 9 (9)

C0428 0 (10) 0 (10) 10 (10)

C0258 0 (10) 0 (10) 10 (10)

NSS1120 0 (10) 0 (10) 10 (10)

UT128B 0 (9) 0 (9) 9 (9)

Note: Numbers in brackets signify total number of samples analysed

A.17 Trial 2 analysis of bacterial presence in root dip inoculated root samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (6) 0 (6) 6 (6)

C0348 0 (6) 0 (6) 6 (6)

C0103 0 (6) 0 (6) 6 (6)

C0428 0 (6) 0 (6) 6 (6)

C0258 0 (6) 0 (6) 6 (6)

NSS1120 0 (6) 0 (6) 6 (6)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

Table A.18 Trial 2 analysis of bacterial presence in root dip inoculated stem samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (6) 0 (6) 6 (6)

C0348 0 (6) 0 (6) 6 (6)

C0103 0 (6) 0 (6) 6 (6)

C0428 0 (6) 0 (6) 6 (6)

C0258 0 (6) 0 (6) 6 (6)

NSS1120 0 (6) 0 (6) 6 (6)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

A.19 Trial 2 analysis of bacterial presence in root dip inoculated leaf samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (6) 0 (6) 6 (6)

C0348 0 (6) 0 (6) 6 (6)

C0103 0 (6) 0 (6) 6 (6)

C0428 0 (6) 0 (6) 6 (6)

C0258 0 (6) 0 (6) 6 (6)

NSS1120 0 (6) 0 (6) 6 (6)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

A.20 Trial 2 analysis of bacterial presence in root dip inoculated control root samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (3) 0 (3) 3 (3)

C0348 0 (4) 0 (4) 4 (4)

C0103 0 (4) 0 (4) 4 (4)

C0428 0 (3) 0 (3) 3 (3)

C0258 0 (4) 0 (4) 4 (4)

NSS1120 0 (4) 0 (4) 4 (4)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

A.21 Trial 2 analysis of bacterial presence in root dip inoculated control stem samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (3) 0 (3) 3 (3)

C0348 0 (4) 0 (4) 4 (4)

C0103 0 (4) 0 (4) 4 (4)

C0428 0 (3) 0 (3) 3 (3)

C0258 0 (4) 0 (4) 4 (4)

NSS1120 0 (4) 0 (4) 4 (4)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

A.22 Trial 2 analysis of bacterial presence in root dip inoculated control leaf samples of greenhouse corn via PCR Genotype PCR Round 1 PCR Round 2 Ubiquitin

C0444 0 (3) 0 (3) 3 (3)

C0348 0 (4) 0 (4) 4 (4)

C0103 0 (4) 0 (4) 4 (4)

C0428 0 (3) 0 (3) 3 (3)

C0258 0 (4) 0 (4) 4 (4)

NSS1120 0 (4) 0 (4) 4 (4)

UT128B 0 (4) 0 (4) 4 (4)

Note: Numbers in brackets signify total number of samples analysed

Pal5 Round 1

Pal5

Round 2

Ubiquitin

A.23 Root tissue PCR analysis of root dip inoculated sorghum genotype N111 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – positive control (Pal5 102 dilution), lanes 14-15 – negative control, lane 16 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – negative control, lane 14 – 100 bp ladder.

Pal5 Round 1

Pal5

Round 2

Ubiquitin

A.24 Stem tissue PCR analysis of root dip inoculated sorghum genotype N111 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – positive control (Pal5 102 dilution), lanes 14-15 – negative control, lane 16 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – negative control, lane 14 – 100 bp ladder.

Pal5

Round 1

Pal5

Round 2

Ubiquitin

A.25 Leaf tissue PCR analysis of root dip inoculated sorghum genotype N111 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – positive control (Pal5 102 dilution), lanes 14-15 – negative control, lane 16 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-7 – inoculated plant tissue samples, lanes 8-11 – un-inoculated plant tissue samples, lanes 12-13 – negative control, lane 14 – 100 bp ladder.

Pal5

Round 1

Pal5

Round 2

Ubiquitin

A.26 PCR analysis of aseptically inoculated corn genotype C0103 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – positive control (Pal5 102 dilution), lanes 18-19 – negative control, lane 20 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – negative control, lane 18 – 100 bp ladder.

Pal5 Round 1

Pal5

Round 2

Ubiquitin

A.27 PCR analysis of aseptically inoculated corn genotype C0348 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – positive control (Pal5 102 dilution), lanes 18-19 – negative control, lane 20 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – negative control, lane 18 – 100 bp ladder.

Pal5 Round 1

Pal5 Round 2

Ubiquitin

A.28 PCR analysis of aseptically inoculated corn genotype C0444 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – positive control (Pal5 102 dilution), lanes 18-19 – negative control, lane 20 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – negative control, lane 18 – 100 bp ladder.

Pal5 Round 1

Pal5 Round 2

Ubiquitin

A.29 PCR analysis of aseptically inoculated corn genotype C0258 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – positive control (Pal5 102 dilution), lanes 18-19 – negative control, lane 20 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – negative control, lane 18 – 100 bp ladder.

Pal5 Round 1

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Ubiquitin

A.30 PCR analysis of aseptically inoculated corn genotype C0428 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – positive control (Pal5 102 dilution), lanes 18-19 – negative control, lane 20 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – negative control, lane 18 – 100 bp ladder.

Pal5 Round 1

Pal5

Round 2

Ubiquitin

A.31 PCR analysis of aseptically inoculated corn genotype NSS120 In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – positive control (Pal5 102 dilution), lanes 18-19 – negative control, lane 20 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-11 – inoculated plant tissue samples, lanes 12-15 – un-inoculated plant tissue samples, lanes 16-17 – negative control, lane 18 – 100 bp ladder.

Pal5 Round 1

Pal5 Round 2

Ubiquitin

A.32 PCR analysis of aseptically inoculated corn genotype UT128B In images Round 1 and Round 2: Lane 1 – 100 bp ladder, lanes 2-10 – inoculated plant tissue samples, lanes 11-14 – un-inoculated plant tissue samples, lanes 15-16 – positive control (Pal5 102 dilution), lanes 17-18 – negative control, lane 19 – 100 bp ladder. In ubiquitin image: Lane 1 – 100 bp ladder, lanes 2-10 – inoculated plant tissue samples, lanes 11-14 – un-inoculated plant tissue samples, lanes 15-16 – negative control, lane 17 – 100 bp ladder.

Curriculum Vitae

Name: Nikita Eskin

Education: 2009-2012 M.Sc. – Biology University of Western Ontario, London, Ontario, Canada

2004-2009 Honours B.Sc. – Biology Minor – Microbiology and Immunology University of Western Ontario, London, Ontario, Canada

Awards/ Honours: 2010-2011 Natural Science and Engineering Research Council Industrial Postgraduate Scholarship 2010-2011 Nominated for the Graduate Student Teaching Award 2009-2011 Western Graduate Research Scholarship 2009 Western Graduate Scholarship 2008-2009 Deans Honour List

Related Work Experience: 2009-2012 Teaching Assistant – First Year Biology Labs University of Western Ontario, London, Ontario, Canada